Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B

Abstract

Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle triglycerides and serum adipokines are also affected similarly by HFD in both groups. Nevertheless, muscle-specific PTP1B(-/-) mice exhibit increased muscle glucose uptake, improved systemic insulin sensitivity, and enhanced glucose tolerance. These findings correlate with and are most likely caused by increased phosphorylation of the insulin receptor and its downstream signaling components. Thus, muscle PTP1B plays a major role in regulating insulin action and glucose homeostasis, independent of adiposity. In addition, rosiglitazone treatment of HFD-fed control and muscle-specific PTP1B(-/-) mice revealed that rosiglitazone acts additively with PTP1B deletion. Therefore, combining PTP1B inhibition with thiazolidinediones should be more effective than either alone for treating insulin-resistant states.

Figures

FIG. 1.

Muscle-specific PTP1B deletion. (a) Deletion efficiency in the indicated tissues from MCK PTP1B−/− and PTP1Bflox/flox littermates. (b) Hematoxylin- and eosin-stained muscles from MCK PTP1B−/− and PTP1Bflox/flox littermates.

FIG. 2.

Muscle-specific PTP1B deletion has no effect on body weight or adiposity. (a) Weight curves for MCK PTP1B−/− (n = 9) and PTP1B+/− (n = 6) mice versus PTP1Bflox/flox controls (n = 9) on chow diet. (b) Weight curves for MCK PTP1B−/− (n = 12) and PTP1B+/− (n = 9) mice versus PTP1Bflox/flox controls (n = 12) on HFD. The curves show similar results to those we obtained previously (18). (c) Fat pad weights of MCK PTP1B−/− (n = 12) and PTP1B+/− (n = 9) mice versus PTP1Bflox/flox mice (n = 12) on HFD. Epi, epididymal; SubQ, subcutaneous; RP, retroperitoneal; Mes, mesenteric; BAT, brown adipose tissue. (d) Total body weight/total body triglyceride ratios from carcass analysis of MCK PTP1B−/− and PTP1Bflox/flox mice on HFD. (e) DEXA analysis of MCK PTP1B−/− (n = 13), MCK PTP1B+/− (n = 6), and PTP1Bflox/flox (n = 14) mice fed a chow diet. Statistical analysis was performed using two-way ANOVA or one-way ANOVA followed by Tukey's multiple comparison test.

FIG. 3.

Improved insulin sensitivity and glucose homeostasis in MCK PTP1B−/− mice. (a) GTT results for male MCK PTP1B−/− (n = 12), MCK PTP1B+/− (n = 6), and PTP1Bflox/flox control (n = 14) mice on chow diet. (b) GTT results for male MCK PTP1B−/− (n = 12), MCK PTP1B+/− (n = 9), and PTP1Bflox/flox (n = 12) mice on HFD. (c) Insulin concentrations during GTTs in male MCK PTP1B−/− (n = 4) and PTP1Bflox/flox (n = 6) mice on HFD. (d) ITT results for male MCK PTP1B−/− (n = 12), MCK PTP1B+/− (n = 6), and PTP1Bflox/flox (n = 14) mice on chow (insulin dose, 0.75 mU/g). (e) ITT results for male MCK PTP1B−/− (n = 12), MCK PTP1B+/− (n = 9), and PTP1Bflox/flox (n = 12) mice on HFD (insulin dose, 1 mU/g). Statistical analysis was performed using two-way ANOVA (**, P < 0.01; *, P < 0.05 [for MCK PTP1B−/− versus PTP1Bflox/flox mice]; ##, P < 0.01; #, P < 0.05 [for MCK PTP1B+/− versus PTP1Bflox/flox mice]).

FIG. 4.

Glucose metabolism as measured by hyperinsulinemic-euglycemic clamp studies. (a) Basal and clamp glucose levels in MCK PTP1B−/− (n = 7) and PTP1Bflox/flox control (n = 5) mice on HFD for 7 weeks. (b) Whole-body glucose infusion rates. (c) Glucose turnover rates. (d) Glycogen synthesis rates. (e) Glucose uptake into gastrocnemius muscle. (f) Glucose uptake into WAT. (g) Hepatic glucose production. The data shown in panels b to f were all measured during the clamp. Results are means with SEM, and statistical analysis was performed using one-tailed Student's t test (**, P < 0.01; *, P < 0.05).

FIG. 5.

Enhanced insulin sensitivity in MCK PTP1B−/− mice. (a and b) IR phosphorylation in muscles from mice fed with chow (a) or HFD (b) and injected with saline or insulin (10 mU/g i.p.). Lysates were immunoprecipitated with IR antibodies, immunoblotted with antiphosphotyrosine antibody, and then stripped and reprobed with IR antibodies to control for loading. Immunoblots were then quantified using an Odyssey infrared imaging system. Bar graphs represent pooled, normalized data (arbitrary units) for MCK PTP1B−/− (n = 4 and 6 [for saline and insulin groups, respectively]) and PTP1Bflox/flox (n = 4 and 6) mice (males; 18 weeks on each diet). (c) IRS1 tyrosine phosphorylation in muscles from HFD-fed MCK PTP1B−/− mice (n = 4 and 6) and PTP1Bflox/flox controls (n = 4 and 6). Lysates were immunoprecipitated with IRS1 antibodies, immunoblotted with antiphosphotyrosine antibody, and then stripped and reprobed with IRS1 antibodies to control for loading. Immunoblots were then quantified using an Odyssey infrared imaging system. (d) IRS1-associated PI3K activity, expressed as x-fold increases from basal activity, in muscles from HFD-fed MCK PTP1B−/− (n = 3 and 6) and PTP1Bflox/flox (n = 3 and 6) mice. There was no difference in basal PI3K activity between MCK PTP1B−/− and PTP1Bflox/flox mice. (e) Akt phosphorylation, as assessed by immunoblots, in muscle lysates from HFD-fed MCK PTP1B−/− (n = 4 and 4) and PTP1Bflox/flox (n = 4 and 4) mice. A representative blot is shown, wherein each lane represents the muscle lysate from a different mouse. The blot was reprobed with anti-PTP1B antibodies to show the absence of PTP1B in muscles of MCK PTP1B−/− mice. Proteins were visualized by enhanced chemiluminescence, and the data were pooled, normalized, and represented in a bar graph. (f) Representative blots of IR phosphorylation in muscle lysates from mice on chow and HFD, as well as IRS1 phosphorylation. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison test (**, P < 0.01; *, P < 0.05).

FIG. 6.

Tissue triglycerides and adipokine levels in MCK PTP1B−/− and PTP1Bflox/flox control mice. (a) Muscle triglycerides in MCK PTP1B−/− (n = 6) and PTP1Bflox/flox control (n = 6) mice on chow and in MCK PTP1B−/− (n = 5) and PTP1Bflox/flox control (n = 5) mice on HFD. (b) Heart triglycerides in MCK PTP1B−/− (n = 6) and PTP1Bflox/flox control (n = 6) mice on chow and in MCK PTP1B−/− (n = 5) and PTP1Bflox/flox control (n = 5) mice on HFD. Serum leptin (c), RBP4 (d), adiponectin (e), and resistin (f) levels in MCK PTP1B−/− (n = 8) and PTP1Bflox/flox control (n = 8) mice on HFD were also measured. Results are means with SEM, and statistical analysis was performed using one-tailed Student's t test (*, P < 0.05).

FIG. 7.

Rosiglitazone treatment leads to improved insulin sensitivity and glucose homeostasis in MCK PTP1B−/− mice. (a) GTT results for male MCK PTP1B−/− (n = 7; filled circles) and PTP1Bflox/flox control (n = 10; filled squares) mice on HFD prior to rosiglitazone treatment and for MCK PTP1B−/− (n = 7; empty circles) and PTP1Bflox/flox (n = 10; empty squares) mice on HFD after rosiglitazone treatment. (b) ITT results for male MCK PTP1B−/− (n = 7; filled circles) and PTP1Bflox/flox (n = 10; filled squares) mice on HFD prior to rosiglitazone treatment (insulin dose, 1.2 mU/g) and for MCK PTP1B−/− (n = 7; empty circles) and PTP1Bflox/flox (n = 10; empty squares) mice on HFD after rosiglitazone treatment (insulin dose, 1.2 mU/g). (c) Fasted (5 h) blood glucose and serum insulin levels before and after rosiglitazone treatment. Statistical analysis was performed using two-way ANOVA (**, P < 0.01; *, P < 0.05 [for MCK PTP1B−/− versus PTP1Bflox/flox mice, both prior to rosiglitazone treatment]; ##, P < 0.01; #, P < 0.05 [for MCK PTP1B−/− versus PTP1Bflox/flox mice, both after rosiglitazone treatment]).