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. 2011 Sep 1;438(2):369-78.
doi: 10.1042/BJ20110373.

Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress

Abdelali Agouni  1 , Nimesh Mody, Carl Owen, Alicja Czopek, Derek Zimmer, Mohamed Bentires-Alj, Kendra K Bence, Mirela Delibegović
Affiliations

Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically induced endoplasmic reticulum stress

Abdelali Agouni et al. Biochem J. .

Abstract

Obesity is associated with induction of the ER (endoplasmic reticulum)-stress response signalling and insulin resistance. PTP1B (protein tyrosine phosphatase 1B) is a major regulator of adiposity and insulin sensitivity. The aim of the present study was to investigate the role of L-PTP1B (liver-specific PTP1B) in chronically HFD (high-fat diet) and pharmacologically induced (tunicamycin and thapsigargin) ER-stress response signalling in vitro and in vivo. We assessed the effects of ER-stress response induction on hepatic PTP1B expression, and consequences of hepatic-PTP1B deficiency, in cells and mouse liver, on components of ER-stress response signalling. We found that PTP1B protein and mRNA expression levels were up-regulated in response to acute and/or chronic ER stress, in vitro and in vivo. Silencing PTP1B in hepatic cell lines or mouse liver (L-PTP1B(-/-)) protected against induction of pharmacologically induced and/or obesity-induced ER stress. The HFD-induced increase in CHOP (CCAAT/enhancer-binding protein homologous protein) and BIP (binding immunoglobulin protein) mRNA levels were partially inhibited, whereas ATF4 (activated transcription factor 4), GADD34 (growth-arrest and DNA-damage-inducible protein 34), GRP94 (glucose-regulated protein 94), ERDJ4 (ER-localized DnaJ homologue) mRNAs and ATF6 protein cleavage were completely suppressed in L-PTP1B(-/-) mice relative to control littermates. L-PTP1B(-/-) mice also had increased nuclear translocation of spliced XBP-1 (X box-binding protein-1) via increased p85α binding. We demonstrate that the ER-stress response and L-PTP1B expression are interlinked in obesity- and pharmacologically induced ER stress and this may be one of the mechanisms behind improved insulin sensitivity and lower lipid accumulation in L-PTP1B(-/-) mice.

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Figures

Figure 1
Figure 1. ER stress induction leads to increased PTP1B expression
(A) Effect of tunicamycin-induced ER stress on PTP1B mRNA expression in HepG2 cells treated with PTP1B- or control-siRNA (*P <0.05, **P<0.01 vs. the indicated group). (B) Effect of tunicamycin-induced ER stress on PTP1B protein levels expression in HepG2 cells treated with PTP1B- or control-siRNA. (C) PTP1B protein levels in livers from control mice fed a chow diet, mice fed a chow diet and injected with tunicamycin and mice fed HFD. Bar graphs represent pooled, normalized data to total amount of SHP2 protein expressed as arbitrary units (n = 3–4 per group). (D) PTP1B mRNA expression in livers from mice injected either with saline or tunicamycin. (E) PTP1B mRNA expression in livers from mice fed chow and HFD (n=3-4 per group) (*P<0.05, **P<0.01 vs. chow or saline). SHP2 (F) and PTEN (G) protein levels in livers from control mice fed a chow diet, mice fed a chow diet and injected with tunicamycin and mice fed HFD. Bar graphs represent pooled, normalized data to total amount of GAPDH protein expressed as arbitrary units (n = 3–4 per group). Data (A-G) were analyzed by t-test or one-way ANOVA, followed by a Tukey’s multiple comparison test.
Figure 2
Figure 2. PTP1B knockdown protects against pharmacologically-induced ER stress in HepG2
Effect of tunicamycin-induced ER stress on eIF2-α phosphorylation (A), XBP-1 splicing (B), and CHOP protein levels (C) in HepG2 cells treated or not with PTP1B siRNA. (D-K) Effect of tunicamycin-induced ER stress on mRNA expression CHOP (D), ATF4 (E), GADD34 (F), BIP (G), GRP94 (H), ERDJ4 (I) in HepG2 cells treated or not with PTP1B siRNA. (J and K) Effect of thapsigargin-induced ER stress on mRNA expression CHOP (J) and ATF4 (K) in HepG2 cells treated either with empty vector or specific PTP1B shRNA (*P<0.05, **P<0.01, ***P<0.001 vs. CTL siRNA or empty vector; #P< 0.05, ## P< 0.01, ### P<0.001 vs. CTL siRNA or empty vector +tunicamycin). Data (B, and D-K) are expressed as mean±SEM and were analyzed two-way ANOVA, followed by a Bonferroni’s multiple comparison test (n =3-7 per group).
Figure 3
Figure 3. Liver PTP1B deletion improves glucose homeostasis
(A) Blood glucose from fl/fl (n =11) and L-PTP1B−/− (n = 7) either fasted overnight or fasted overnight then re-fed for 1h (*P<0.05, ***P<0.001 vs. the indicated group). (B) Circulating insulin from fl/fl and L-PTP1B−/− either fasted overnight or fasted overnight then re-fed for 1h (**P<0.01, ***P<0.001 vs. the indicated group). (C) Homeostatic model assessment (HOMA) calculated for fl/fl and L-PTP1B−/− either fasted overnight or fasted overnight then re-fed for 1h (*P<0.05, **P<0.01 vs. the indicated group). (D-H) Relative mRNA expression, measured by quantitative real-time PCR normalized against β-actin mRNA, of SREBP-1C (D), FAS (E), PPAR-γ (F), HMGCS (G) and PPAR-α (H) in livers of male fl/fl and L-PTP1B−/− mice fed chow or HFD (n=6-13) (*P<0.05, **P<0.01, ***P<0.001 vs. chow fl/fl; #P<0.05 vs. HFD fl/fl). Data (A-J) are expressed as mean±SEM and were analyzed by two-way ANOVA, followed by a Bonferroni’s multiple comparison test.
Figure 4
Figure 4. Liver PTP1B-deficiency decreases ER stress response regulated through PERK/eIF2-α pathway
(A) eIF2-α phosphorylation on S51 in livers of male fl/fl and L-PTP1B−/− mice on chow or HFD (n=3-6 per group). (B) CHOP protein expression in livers from male fl/fl and L-PTP1B−/− mice fed chow or HFD. Bar graphs represent pooled, normalized data to total amount of ERK1 protein expressed as arbitrary units (n =4 per group) (*P < 0.05, **P < 0.01 vs. chow fl/fl; #P < 0.05 vs. HFD fl/fl). (C-E) mRNA expression of CHOP (C), ATF4 (D) and GADD34 (E) in livers of male fl/fl and L-PTP1B−/− mice fed chow or HFD (n=6-13 per group) (*P<0.05, **P<0.01 vs. chow fl/fl; #P<0.05 vs. HFD fl/fl). Data (B-E) are expressed as mean±SEM and were analyzed two-way ANOVA, followed by a Bonferroni’s multiple comparison test.
Figure 5
Figure 5. Liver PTP1B-deficiency decreases ER stress response regulated through ATF6 pathway
ATF6 protein expression in livers from male fl/fl and L-PTP1B−/− mice fed chow or HFD. Bar graphs represent pooled, normalized data to total amount of ERK1 protein from expressed as arbitrary units (n=4 per group) of full length and cleaved forms of ATF6 (*P<0.05 vs. chow fl/fl; **P<0.01 vs. the indicated group; ##P<0.01 vs. HFD L-PTP1B−/−). Data are expressed as mean±SEM and were analyzed two-way ANOVA, followed by a Bonferroni’s multiple comparison test.
Figure 6
Figure 6. Liver PTP1B-deficiency decreases ER stress response regulated through IRE1-α sensor and increases XBP-1 nuclear translocation through p85-α interaction
(A) IRE1-α mRNA expression in livers from male fl/fl and L-PTP1B−/− mice fed chow or HFD (n =6-13 per group) (*P<0.05 vs. chow fl/fl). (B) IRE1-α phosphorylation on S724 in livers of male fl/fl and L-PTP1B−/− mice on HFD (n=4 per group). (C) Spliced XBP-1 protein expression in livers from male fl/fl and L-PTP1B−/− mice fed chow or HFD (n =3-5 per group) (*P<0.05 vs. chow fl/fl). (D) Spliced XBP-1 protein expression in livers from male fl/fl and L-PTP1B−/− mice injected either with saline or tunicamycin (n =3-4 per group) (*P < 0.05 vs. chow fl/fl; #P<0.05 vs. HFD fl/fl). (E) BIP protein expression in livers from male fl/fl and L-PTP1B−/− mice fed chow or HFD (n =4 per group) (*P<0.05 vs. chow fl/fl; #P<0.05 vs. HFD fl/fl). BIP and CHOP (Figure 2B) were blotted on the same gel; hence they share the same ERK1 control blot. Bar graphs represent pooled, normalized data to total amount of ERK1 or ERK2 protein expressed as arbitrary units. (F-H) mRNA expression of EDEM1 (F), ERDJ4 (G) and GRP94 (H) in livers of male fl/fl and L-PTP1B−/− mice fed chow or HFD (n =6-13 per group) (*P<0.05, **P<0.01 vs. chow fl/fl; #P<0.05 vs. HFD fl/fl). Data (A-H) were analyzed by two-way ANOVA, followed by a Bonferroni’s multiple comparison test. (I) XBP-1 was immunoprecipitated from nuclear and cytosolic liver lysates (fl/fl and L-PTP1B−/− mice on HFD) and immunoblotted for the p85-α regulatory subunit of phosphatidylinositol 3-kinase and spliced XBP-1 (n =3 per group). (J) Total p85-α protein levels in livers from fl/fl and L-PTP1B−/− mice fed HFD (n =4 per group). (K) To assess the purity of nuclear and cytosolic fractions we blotted for a nuclear marker, lamin A/C, and for ERK1 as total.
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