Rosiglitazone ameliorates abnormal expression and activity of protein tyrosine phosphatase 1B in the skeletal muscle of fat-fed, streptozotocin-treated diabetic rats

Abstract

Protein tyrosine phosphatase 1B (PTP1B) acts as a physiological negative regulator of insulin signaling by dephosphorylating the activated insulin receptor (IR). Here we examine the role of PTP1B in the insulin-sensitizing action of rosiglitazone (RSG) in skeletal muscle and liver. Fat-fed, streptozotocin-treated rats (10-week-old), an animal model of type II diabetes, and age-matched, nondiabetic controls were treated with RSG (10 micromol kg(-1) day(-1)) for 2 weeks. After RSG treatment, the diabetic rats showed a significant decrease in blood glucose and improved insulin sensitivity. Diabetic rats showed significantly increased levels and activities of PTP1B in the skeletal muscle (1.6- and 2-fold, respectively) and liver (1.7- and 1.8-fold, respectively), thus diminishing insulin signaling in the target tissues. We found that the decreases in insulin-stimulated glucose uptake (55%), tyrosine phosphorylation of IRbeta-subunits (48%), and IR substrate-1 (IRS-1) (39%) in muscles of diabetic rats were normalized after RSG treatment. These effects were associated with 34 and 30% decreases in increased PTP1B levels and activities, respectively, in skeletal muscles of diabetic rats. In contrast, RSG did not affect the increased PTP1B levels and activities or the already reduced insulin-stimulated glycogen synthesis and tyrosine phosphorylation of IRbeta-subunits and IRS-2 in livers of diabetic rats. RSG treatment in normal rats did not significantly change PTP1B activities and levels or protein levels of IRbeta, IRS-1, and -2 in diabetic rats. These data suggest that RSG enhances insulin activity in skeletal muscle of diabetic rats possibly by ameliorating abnormal levels and activities of PTP1B.

Figure 1

Effects of RSG on glucose uptake into skeletal muscle from control and HFD/STZ rats. Epitrochlearis muscles from saline- and RSG-treated animals were removed and incubated in the presence or absence of 12 nM insulin. 2-DOG uptake was measured as described in Methods. Values are the means±s.e.m. of six independent experiments. ^*P<0.05 basal vs insulin-stimulated conditions; ^¶P<0.05 vs HFD/STZ-untreated rats.

Figure 2

Effects of RSG on glycogen synthesis in liver from control and HFD/STZ rats. Hepatocytes were incubated for 3 h in minimal essential medium containing [U-¹⁴C]glucose (2 μCi ml⁻¹) with or without insulin (10 nM). Incorporation of ¹⁴C label into glycogen was determined by ethanol precipitation, and rates of glycogen synthesis are expressed as nmol glucose incorporated per 3 h per mg cell protein. Values represent the means±s.e.m. for six independent experiments. ^*P<0.05 basal vs insulin-stimulated conditions; ^¶P<0.05 vs insulin-stimulated control groups.

Figure 3

Effects of RSG on PTP1B protein expression in skeletal muscle and liver from control and HFD/STZ rats. Skeletal muscle and liver tissue samples were obtained from the different groups. After lysate preparation, equal amounts of muscle (a) or liver protein (b) underwent SDS–PAGE and were subjected to Western blotting with specific antibodies against PTP1B (α-PTP1B). Immunoreactive bands for PTP1B were identified using ECL, were scanned and normalized to β-actin. The control was set to 100% and the different groups are expressed as a percentage of control levels (c, d). Data are presented as the means±s.e.m. of six independent experiments. ^*P<0.05 vs RSG-untreated control rats. ^¶P<0.05 vs RSG-untreated HFD/STZ rats.

Figure 4

Effects of RSG on PTP1B activity in muscle (a) and liver (b) from control and HFD/STZ rats. The muscle and liver tissue homogenate was assayed in a microtiter plate at 27°C. PTP1B activity was measured using a PTP1B assay kit. Means±s.e.m. of six independent experiments is expressed as relative to normal values, which were set as 100%. ^**P<0.01 vs RSG-untreated control rats. ^¶P<0.05 vs RSG-untreated HFD/STZ rats.

Figure 5

Effects of RSG on insulin-induced tyrosine phosphorylation of the IR β-subunit. A sample of muscle (left panel) or liver (right panel) tissue was obtained following injection with insulin (+) or its diluent (−). Equal amounts of solubilized protein were immunoprecipitated (IP) with an anti-IR-β antibody, followed by SDS–PAGE, and were analyzed by Western blotting with PY99 (a, e). The blots were stripped and reprobed with IR-β (b, f). Phosphorylation (c, g) and expression levels (d, h) of IR-β were quantified by densitometry and expressed relative to control (basal) samples. Error bars represent the s.e.m. of six independent experiments. ^*P<0.05 vs RSG-untreated control rats. ^¶P<0.05 vs RSG-untreated HFD/STZ rats.

Figure 6

Effects of RSG on insulin-induced tyrosine phosphorylation of IRS-1 and -2. A sample of muscle (left panel) of liver (right panel) tissue was obtained following injection with insulin (+) or its diluent (−). Equal amounts of solubilized protein were immunoprecipitated (IP) with an anti-IRS-1 or anti-IRS-2 antibody, followed by SDS–PAGE, and were analyzed by Western blotting with PY99. (a, e). The blots were stripped and reprobed with IRS-1 (b) and IRS-2 (f). Phosphorylation (c, g) and expression levels (d, h) of IRS-1 and -2 were quantified by densitometry and expressed relative to control (basal) samples. Error bars represent s.e.m. of six independent experiments. ^*P<0.05 vs RSG-untreated control rats. ^¶P<0.05 vs RSG-untreated HFD/STZ rats.