Liver-specific deletion of protein-tyrosine phosphatase 1B (PTP1B) improves metabolic syndrome and attenuates diet-induced endoplasmic reticulum stress

Abstract

Objective: The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling; consequently, mice deficient in PTP1B are hypersensitive to insulin. Because PTP1B(-/-) mice have diminished fat stores, the extent to which PTP1B directly regulates glucose homeostasis is unclear. Previously, we showed that brain-specific PTP1B(-/-) mice are protected against high-fat diet-induced obesity and glucose intolerance, whereas muscle-specific PTP1B(-/-) mice have increased insulin sensitivity independent of changes in adiposity. Here we studied the role of liver PTP1B in glucose homeostasis and lipid metabolism.

Research design and methods: We analyzed body mass/adiposity, insulin sensitivity, glucose tolerance, and lipid metabolism in liver-specific PTP1B(-/-) and PTP1Bfl/fl control mice, fed a chow or high-fat diet.

Results: Compared with normal littermates, liver-specific PTP1B(-/-) mice exhibit improved glucose homeostasis and lipid profiles, independent of changes in adiposity. Liver-specific PTP1B(-/-) mice have increased hepatic insulin signaling, decreased expression of gluconeogenic genes PEPCK and G-6-Pase, enhanced insulin-induced suppression of hepatic glucose production, and improved glucose tolerance. Liver-specific PTP1B(-/-) mice exhibit decreased triglyceride and cholesterol levels and diminished expression of lipogenic genes SREBPs, FAS, and ACC. Liver-specific PTP1B deletion also protects against high-fat diet-induced endoplasmic reticulum stress response in vivo, as evidenced by decreased phosphorylation of p38MAPK, JNK, PERK, and eIF2alpha and lower expression of the transcription factors C/EBP homologous protein and spliced X box-binding protein 1.

Conclusions: Liver PTP1B plays an important role in glucose and lipid metabolism, independent of alterations in adiposity. Inhibition of PTP1B in peripheral tissues may be useful for the treatment of metabolic syndrome and reduction of cardiovascular risk in addition to diabetes.

FIG. 1.

Liver-specific PTP1B deletion has no effect on body weight or adiposity. A: Deletion efficiency, as detected by PCR and Western blotting, in the indicated tissues from Alb-Cre-PTP1B^−/− and fl/fl littermates. PCR was performed on DNA isolated from Alb-Cre-PTP1B^−/− tissues: P, pituitary; B, brain; L, liver; M, gastrocnemius muscle; S, spleen; H, heart; W, abdominal white adipose tissue; K, kidney; and L, lung. “−” and “+” represent negative and positive controls, respectively. The immunoblot in the lower panel indicates ∼80% deletion of PTP1B protein in liver, consistent with deletion from hepatocytes, which comprise that approximate percentage of the liver. B: Weight curves for Alb-Cre-PTP1B^−/− (n = 6) and Alb-Cre-PTP1B^+/− mice (n = 10) vs. fl/fl controls (n = 16) on chow diet from weaning to 17 weeks of age (left). Weight curves for Alb-Cre-PTP1B^−/− (n = 12) and fl/fl controls (n = 12) on HFD from weaning to 19 weeks of age (right). C: Fat pad weights of 19-week-old Alb-Cre-PTP1B^−/− (n = 12) and Alb-Cre-PTP1B^+/− (n = 9) vs. fl/fl mice (n = 12) on HFD. BAT, brown adipose tissue; EPI, epididymal; MES, mesenteric; RP, retroperitoneal; and SUB Q, subcutaneous. D: Leptin levels (ng/ml) in Alb-Cre-PTP1B^−/− (n = 5) and fl/fl mice (n = 9) on chow diet and Alb-Cre-PTP1B^−/− (n = 10) and fl/fl mice (n = 12) on HFD (at 5 months of age). E: Length (cm) of Alb-Cre-PTP1B^−/− (n = 12) and PTP1B^+/− (n = 9) vs. fl/fl mice (n = 12) on HFD.

FIG. 2.

Improved glucose homeostasis in Alb-Cre-PTP1B^−/− mice. A: Insulin-to-glucose ratio in male Alb-Cre-PTP1B^−/− (n = 13), Alb-Cre PTP1B^+/− (n = 8), and fl/fl (n = 14) mice on HFD at 8 and 16 weeks of age, respectively. B: GTTs on male Alb-Cre-PTP1B^−/− (n = 6), Alb-Cre PTP1B^+/− (n = 6), and fl/fl control mice (n = 8) on chow diet at 8 weeks of age. C: GTTs on male Alb-Cre-PTP1B^−/− (n = 6), Alb-Cre PTP1B^+/− (n = 8), and fl/fl mice (n = 8) on HFD for 8 weeks (11 weeks of age). D: ITTs on male Alb-Cre-PTP1B^−/− (n = 6), Alb-Cre PTP1B^+/− (n = 6), and fl/fl mice (n = 8) on chow diet at 8 weeks of age (insulin dose 0.75 mU/g). E: ITTs on male Alb-Cre-PTP1B^−/− (n = 6) and fl/fl mice (n = 8) on HFD for 7 weeks (10 weeks of age) (insulin dose 1 mU/g). Data were analyzed by two-way ANOVA (*P < 0.05 for Alb-Cre-PTP1B^−/− vs. fl/fl).

FIG. 3.

Hyperinsulinemic-euglycemic clamp studies. Alb-Cre-PTP1B^−/− (n = 15) and fl/fl control (n = 13) mice on chow diet at 18–20 weeks of age were subjected to hyperinsulinemic-euglycemic clamp studies (see research design and methods). A: Hepatic glucose production. B: Hepatic insulin action (percent suppression of hepatic glucose production). C: Basal and clamp blood glucose levels. D: Whole-body glucose infusion rate. E: Whole-body glucose turnover. F: Whole-body glycogen and lipid synthesis. G: Glucose uptake into gastrocnemius muscle. Results are mean ± SE. Data were analyzed by one-tailed Student's t test (*P < 0.05; #P = 0.052).

FIG. 4.

Enhanced insulin sensitivity in Alb-Cre-PTP1B^−/− mice. A and B: Insulin receptor phosphorylation on Y1162/1163 in livers of 18-week-old male mice on chow (A) or HFD (B) and injected with saline or insulin (10 mU/g i.p.). C: IRS-1 tyrosine phosphorylation in liver as measured by ELISA in 18-week-old mice on HFD. D: IRS-2 tyrosine phosphorylation in liver of 18-week-old mice on HFD, quantified by IRS-2 immunoprecipitation and anti-phosphotyrosine immunoblotting. Bar graphs represent pooled, normalized data to total amount of IRS protein (arbitrary units [AU]) from Alb-Cre-PTP1B^−/− and fl/fl mice (n = 6–8 per group). Data (A–D) were analyzed by one-way ANOVA, followed by a Tukey's multiple comparison test (*P < 0.05). E: IRS-1 and IRS-2 were immunoprecipitated from liver lysates (18-week-old mice on HFD) and immunoblotted for the p85 regulatory subunit of phosphatidylinositol 3-kinase. Liver lysates from 18-week-old mice on HFD were also immunoblotted for total levels of p85, phospho-glycogen synthase (GS), and glycogen synthase. F: Relative expression of PGC1, PEPCK, and G-6-Pase mRNAs, normalized against 18S mRNA levels, measured by quantitative real-time PCR in livers from fasted male Alb-Cre-PTP1B^−/− (n = 6) and fl/fl control mice (n = 6) fed HFD for 18 weeks. Results are the means ± SE; data were analyzed using a two-tailed Student's t test (*P < 0.05, **P < 0.01).

FIG. 5.

Improved lipid metabolism in Alb-Cre-PTP1B^−/− mice. Liver triglycerides (A) or serum triglycerides (B) are shown for male Alb-Cre-PTP1B^−/− (n = 8) and control (n = 8) mice fed HFD for the indicated time in weeks. C and D: Relative mRNA levels, measured by quantitative real-time PCR normalized against 18S mRNA, in livers from male Alb-Cre-PTP1B^−/− (n = 8) and fl/fl control mice (n = 8) fed HFD for 15 weeks (18 weeks of age) and fasted overnight. Liver (E) and serum (F) cholesterol in male Alb-Cre-PTP1B^−/− (n = 8) and fl/fl control mice (n = 8) on HFD for 15–17 weeks (18–20 weeks of age) is shown. Results represent the means ± SE. Data were analyzed by two-tailed Student's t test (*P < 0.05, **P < 0.01). ▪, fl/fl; □, Alb-Cre PTP1B^−/−.

FIG. 6.

Alb-Cre-PTP1B^−/− mice are resistant to HFD-induced ER stress. A and B: Liver lysates from mice fed HFD for 18 weeks were analyzed by immunoblotting for p38 MAPK, JNK, PERK, and eIF2α phosphorylation. Blots were stripped and re-probed with total p38 antibodies. JNK, PERK, and eIF2α levels could not be assessed on re-probes, so the same samples were resolved on a parallel gel and immunoblotted for total levels. All immunoblots were quantified by densitometry (Image J). Bar graphs represent pooled data (% difference from control) from Alb-Cre-PTP1B^−/− and fl/fl mice. C: Expression of CHOP mRNA at 5 and 18 weeks HFD (C) XBP1 at 18 weeks HFD (D) were measured by quantitative real-time PCR and normalized to 18S expression in livers from Alb-Cre-PTP1B^−/− (n = 6) and fl/fl control mice (n = 6). Liver lysates from mice fed HFD for 18 weeks were also analyzed by immunoblotting for total levels of CHOP protein, and these blots are included in C. Results represent the means ± SE, and significance was assessed by two-tailed Student's t test (*P < 0.05, **P < 0.01).