PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

Abstract

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

Fig 1.

PTP1B protein levels in liver (A), fat (B), and skeletal muscle (C) from ob/ob mice treated i.p. for 6 weeks twice per week with PTP1B ASO at the indicated dose. (D) Liver PTP1B protein levels in db/db mice treated i.p. once per week for 4 weeks at the indicated dose. In the db/db study, a UC oligonucleotide dosed at 50 mg/kg was included to control for nonspecific effects of the PTP1B ASO. Data are mean ± SE and statistics were determined as a two-tailed t test. *, P < 0. 05; ***, P < 0.001 (vs. saline control).

Fig 2.

Non-fasting plasma glucose (A) and insulin (B) levels versus time in PTP1B ASO-treated ob/ob mice (25, 2.5, and 0.25 mg/kg). Nonfasting plasma glucose (C) levels versus time in PTP1B ASO-treated db/db mice (50, 25, 10, and 50 mg/kg UC). Data are mean ± SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. saline control). In A, the significance of week 3–6 values dosed at 2.5 mg/kg is P < 0.05, and for week 2–6 values dosed at 25 mg/kg is P < 0.01. In B, the significance of week 3–6 values dosed at 25 mg/kg is P < 0.01. In C, the significance of week 2–4 values dosed at 10 mg/kg is P < 0.05, at 25 mg/kg is P < 0.01, and at 50 mg/kg PTP1B ASO is P < 0.001.

Fig 3.

PTP1B ASO treatment increases insulin sensitivity in ob/ob mice. (A) Change in AUC (area under the curve) for plasma glucose after an i.p. GTT. (B) Change in plasma glucose level after an ITT in ob/ob mice. Results are expressed as change from baseline AUC_Glucose for GTT (25, 2.5, and 0.25 mg/kg PTP1B ASO treatment in ob/ob and 25 mg/kg PTP1B ASO treatment in lean littermates, ob/+). Results are expressed as percentage change from baseline for ITT. GTT and ITT were performed in week 6 of treatment. Data are mean ± SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. saline control).

Fig 4.

PTP1B ASO treatment affects the level of IRS-2 and PI3-kinase regulatory subunit (p85α and p50α) expression in ob/ob mouse liver and fat. Representative immunoblots using anti-IRS-2 antibodies (A and B) or anti-p85α whole antiserum that recognized all p85 isoforms (C–F) were quantified. The results are the average of four mice within each group. The data are represented as arbitrary units and are the mean ± SEM. Statistics were determined as a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (vs. saline control).

Fig 5.

PTP1B ASO causes enhanced insulin signaling in liver. PTP1B ASO (25 mg/kg, i.p. dosed twice per week for 6 weeks) or saline-treated ob/ob mice were fasted for 5 h and then challenged with an i.p. bolus of saline or insulin (2 units/kg in 0.1% BSA). Liver tissue was taken at 1 min following saline or insulin challenge. (A) Cell lysates were immunoprecipitated using anti-phosphotyrosine (PY) antibodies and immunoblotted with anti-PY and anti-IR antibodies as indicated. (B) Cell lysates were loaded by equal amount of proteins, separated by SDS/PAGE (7.5% gels), and immunoblotted with anti-phosphothreonine-308-specific PKB antibodies.