Table 1.
Morphometry and fasting glucose of C57BL/6 and PTP1BKO female mice fed with a ND or a HFD for a 20 week period.
Figure 1.
PTP1B knockout mice are protected against obesity-induced glucose intolerance and hepatic steatosis.
Female C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks. At the end of the experiment IPGTT (a) and IPITT (b) were performed. Area under the curve (AUC) for each individual curve of IPGTT (c) and IPITT (d) was calculated (n = 6). Liver fat content was analyzed with (e) Oil Red O staining (n = 6), (f) triglyceride assay kit (n = 6), and (g) cholesterol quantitation colorimetric kit (n = 6). Red staining – fat droplets, blue – DAPI stained nuclei. Scale bars, 100±µm. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice.
Figure 2.
PTP1B deletion improves diet-induced insulin resistance and glucose uptake in skeletal muscle in mice.
(a) [3H]-2-deoxy-glucose-uptake assay (n = 6); (b) representative Western blots and (c) densitometric analysis (n = 6) of Akt phosphorylation in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks and challenged with insulin for 30 minutes. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice.
Figure 3.
The effect of PTP1B deletion on obesity-induced ER stress in skeletal muscle.
Representative Western blots (a), PTP1B activity assay (b) and densitometric analysis (n = 6) of PTP1B (c), GRP78 (d), phospho- and total-eIF2α (p-eIF2α and t-eIF2α, respectively) (e), and phospho- and total-JNK2 (p-JNK and t-JNK, respectively) (f) protein levels in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice.
Figure 4.
The effect of PTP1B deletion on autophagic flux and NCK1 expression in skeletal muscle of obese mice.
Representative Western blots (a) and densitometric analysis (n = 6) of ATG5 (b), ATG7 (c), Beclin-1 (d), p62 (e), and NCK1 (f) protein levels in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 20 weeks. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice, ‡ p<0.05 compared with PTP1BKO ND mice.
Figure 5.
The effect of PTP1B deletion on short term high-fat diet-induced ER stress in skeletal muscle.
Representative Western blots (a) and densitometric analysis (n = 6) of PTP1B (b), GRP78 (c), phospho- and total-eIF2α (p-eIF2α and t-eIF2α, respectively) (d), and phospho- and total-JNK2 (p-JNK and t-JNK, respectively) (e), p62 (f), and NCK1 (g) protein levels in gastrocnemius muscles of the C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) diets for 3 weeks. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice.
Figure 6.
The effect of NAC and TUDCA on tunicamycin (TM)-induced ROS production in cultured myotubes.
Representative confocal microscopy pictures (a) and spectrophotometric quantification (n = 6) (b) of intracellular ROS production in C2C12 myotubes treated with tunicamycin in presence of 10 mmol/l NAC or 1 mmol/l TUDCA for 24 hours. ROS detection dye DHE is shown as red, the nucleus is stained with DAPI (blue), with purple suggests colocalization. Scale bars, 50 µm.
Figure 7.
The role of ROS in tunicamycin (TM)-induced PTP1B protein expression in C2C12 myotubes.
(a–d) Representative Western blots (a) and densitometric analysis (n = 6) of PTP1B (b), GRP78 (c), and phospho- and total-eIF2α (p-eIF2α and t-eIF2α, respectively) (d) protein levels in C2C12 myotubes treated with tunicamycin in presence of 10 mmol/l NAC for 24 hours. (e–g) Representative Western blots (e) and densitometric analysis (n = 6) of GRP78 (f), and p-eIF2α and t-eIF2α (g) protein levels in C2C12 myotubes treated with tunicamycin in presence of 1 mmol/l TUDCA for 24 hours. * p<0.05 compared with non-treated control cells, † p<0.05 compared with tunicamycin-treated cells.
Figure 8.
The role of NFκB in tunicamycin (TM)-induced PTP1B expression in myotubes.
Representative Western blots (a) and densitometric analysis (n = 6) of nuclear NFκB (Nucl. NFκB) (b), cytosolic NFκB (Cyt. NFκB) (c), and PTP1B (d) protein expression in C2C12 myotubes treated with tunicamycin in presence of 10 mmol/l NAC, 100 μmol/l PDTC or 1 mmol/l TUDCA for 24 hours * p<0.05 compared with non-treated control cells, † p<0.05 compared with tunicamycin-treated cells.
Figure 9.
The role of ROS and NFκB in high-fat diet-induced PTP1B expression in skeletal muscle.
Gastrocnemius muscles were isolated from C57 or PTP1BKO mice received normal (ND) or high-fat content (HFD) in a presence or absence of NAC supplementation diets for 3 weeks. (a–b) Representative confocal microscopy pictures (a) and quantification (n = 6) (b) of intracellular ROS production. ROS detection dye DHE is shown as red, the nucleus is stained with DAPI (blue), with purple suggests colocalization. Scale bars, 50 µm. (c–f) Representative Western blots (c) and densitometric analysis (n = 6) of nuclear NFκB (Nucl. NFκB) (d), cytosolic NFκB (Cyt. NFκB) (e), and PTP1B (f) protein expression. * p<0.05 compared with C57 ND mice, † p<0.05 compared with C57 HFD mice.