Journal: The Journal of Biological Chemistry

Article Title: Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes *

doi: 10.1074/jbc.M113.539189

Figure Lengend Snippet: Effect of APAP in JNK activation, IRS1 Ser307 phosphorylation, and PTP1B expression and activity in human primary hepatocytes. A, human primary hepatocytes were treated with APAP (10 mm) for various time periods. GSH content was analyzed at 8 and 12 h. The results are expressed as percentage of untreated cells (100%). Released LDH activity was analyzed at 24 h as described under “Experimental Procedures.” B, phosphorylation of IRS1 (Ser307), JNK1/2, total IRS1, and total JNK1/2 were analyzed by Western blot at early time periods (2–8 h). C, PTP1B, SHP1, and TCPTP mRNA levels (analyzed by RT-PCR), PTP1B protein expression (analyzed by Western blot), and PTP1B enzymatic activity (see “Experimental Procedures”) were determined in primary human hepatocytes treated with 10 mm APAP for 24 h. p85-PI3K (p85) antibody was used as loading control. Representative autoradiograms are shown. The results of PTP1B enzymatic activity are expressed as pmol of released phosphate/min/μg protein. **, p < 0.01 APAP-treated versus untreated hepatocytes (n = 3 independent experiments performed in duplicate).

Article Snippet: Anti-IRS-1 (06-248), anti-IRS2 (06-506), anti-p85-PI3K (06-195), anti-PTP1B (07-088), anti-phospho-IRS1 (Ser 307 ) (07-247), anti-phospho-IRS1 (Tyr 1179 ) (07-844), catalase (219010), and anti-phosphotyrosine (clone 4G10; 05-321) antibodies were from Merck Millipore (Merck).

Techniques: Activation Assay, Expressing, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes *

doi: 10.1074/jbc.M113.539189

Figure Lengend Snippet: Effect of APAP in insulin-mediated signaling in human primary hepatocytes. A, human primary hepatocytes were treated with APAP (10 mm) for 24 h. Then hepatocytes were further stimulated with 10 nm insulin for 10 min, and insulin signaling cascade was analyzed by Western blot with the antibodies against phospho-IR (Tyr1150/1151), phospho-IR (Tyr1162/1163), IR, phospho-IRS1 (Tyr1179), IRS1, IRS2, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt. Immunoprecipitation was performed with anti-IR antibody followed by Western blot with the anti-phosphotyrosine antibody. Representative autoradiograms are shown. B, autoradiograms were quantitated by scanning densitometry. The results are means ± S.E. *, p < 0.05; **, p < 0.01 APAP-treated versus untreated hepatocytes (n = 3 independent experiments performed in duplicate). I.P., immunoprecipitation; W.B., Western blot.

Article Snippet: Anti-IRS-1 (06-248), anti-IRS2 (06-506), anti-p85-PI3K (06-195), anti-PTP1B (07-088), anti-phospho-IRS1 (Ser 307 ) (07-247), anti-phospho-IRS1 (Tyr 1179 ) (07-844), catalase (219010), and anti-phosphotyrosine (clone 4G10; 05-321) antibodies were from Merck Millipore (Merck).

Techniques: Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes *

doi: 10.1074/jbc.M113.539189

Figure Lengend Snippet: Rosiglitazone decreased PTP1B levels and improved insulin sensitivity in APAP-treated human primary hepatocytes. A, human primary hepatocytes were treated with APAP (10 mm) with or without rosiglitazone (20 μm) for 24 h. Total cell lysates were analyzed by Western blot with the antibodies against phospho-JNK1/2, JNK1/2, PTP1B, and p85-PI3K (p85) as a loading control. Autoradiograms corresponding to PTP1B levels were quantitated by scanning densitometry. The results are means ± S.E. ***, p < 0.001 rosiglitazone-treated versus nontreated cells (n = 3 independent experiments performed in duplicate). B, after APAP and rosiglitazone treatment, hepatocytes were stimulated with 10 nm insulin for 10 min. Insulin signaling cascade was analyzed by Western blot with the antibodies against phospho-IR (Tyr1162/Tyr1163), IR, phospho-IRS1 (Tyr1179), IRS1, IRS2, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt. Representative autoradiograms of four independent experiments are shown. Rosi, rosiglitazone.

Article Snippet: Anti-IRS-1 (06-248), anti-IRS2 (06-506), anti-p85-PI3K (06-195), anti-PTP1B (07-088), anti-phospho-IRS1 (Ser 307 ) (07-247), anti-phospho-IRS1 (Tyr 1179 ) (07-844), catalase (219010), and anti-phosphotyrosine (clone 4G10; 05-321) antibodies were from Merck Millipore (Merck).

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes *

doi: 10.1074/jbc.M113.539189

Figure Lengend Snippet: Reduction of PTP1B levels in human Huh7 cell line protected against APAP-mediated inhibition of insulin signaling. A, Huh7 human hepatoma cells were treated with APAP (5 mm) for 24 h. Shown are representative phase contrast microscopy images (scale bar represents 100 μm), GSH content at 8 and 12 h, and released LDH activity at 24 h of APAP treatment. B, Huh7 cells were treated with APAP (5 mm) for various time periods (2–4 h). Total cell lysates were analyzed by Western blot with the antibodies against phospho-IRS1 (Ser307), IRS1, phospho-JNK1/2, and JNK1/2. Representative autoradiograms are shown. PTP1B protein levels were analyzed at 24 h of APAP (5 mm) treatment. p85-PI3K (p85) was used as a loading control. C, Huh7 cells were infected with lentivirus bearing scrambled or PTP1B shRNA (right panels) or transfected with scrambled or PTP1B siRNA oligonucleotides (left panels). Total cell lysates were analyzed by Western blot with the corresponding antibodies against PTP1B and p85-PI3K (p85) as a loading control (n = 3 independent experiments). Cells were stimulated with insulin (1–10 nm) for 10 min, and insulin signaling cascade was analyzed by Western blot with the antibodies against phospho-IR (Tyr1162/1163), IR, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt. Representative autoradiograms are shown (n = 3 independent experiments). D, Huh7 cells, infected with control and PTP1B shRNA lentiviral particles, were treated with 5 mm APAP for 24 h or left untreated. Then hepatocytes were stimulated with insulin (10 nm) for 10 min, and insulin signaling cascade was analyzed by Western blot with the antibodies against phospho-IR (Tyr1162/1163), IR, phospho-IRS1 (Tyr1179), IRS1, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt. Representative autoradiograms are shown (n = 3 independent experiments).

Article Snippet: Anti-IRS-1 (06-248), anti-IRS2 (06-506), anti-p85-PI3K (06-195), anti-PTP1B (07-088), anti-phospho-IRS1 (Ser 307 ) (07-247), anti-phospho-IRS1 (Tyr 1179 ) (07-844), catalase (219010), and anti-phosphotyrosine (clone 4G10; 05-321) antibodies were from Merck Millipore (Merck).

Techniques: Inhibition, Microscopy, Activity Assay, Western Blot, Infection, shRNA, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes *

doi: 10.1074/jbc.M113.539189

Figure Lengend Snippet: PTP1B-deficient hepatocytes are protected against APAP-mediated effects on insulin signaling. A, wild-type (PTP1B+/+) (left panel) or PTP1B−/− (right panel) immortalized mouse hepatocytes were treated with APAP (0.5 mm) for 24 h. Then hepatocytes were stimulated with insulin (10 nm) for 10 min. IR, IRS1, and IRS2 tyrosine phosphorylation were analyzed by immunoprecipitation with the corresponding antibodies followed by Western blot with the anti-phosphotyrosine antibody. Total IR, IRS1, and IRS2 were analyzed in whole cell lysates. B, phosphorylation of Akt was analyzed by Western blot with the antibodies against phospho-Akt (Ser473), phospho-Akt (Thr308), and total Akt. Representative autoradiograms are shown (n = 3 independent experiments). C, wild-type (PTP1B+/+) and PTP1B−/− immortalized mouse hepatocytes were treated with APAP (0.5 mm) for various time periods. IRS1 (Ser307) phosphorylation was analyzed by immunoprecipitation with the anti-IRS1 antibody followed by Western blot with the anti-phospho-IRS1 (Ser307) antibody. Total cell lysates were analyzed by Western blot with the antibodies against IRS1, phospho-JNK1/2, and JNK1/2. Representative autoradiograms are shown (n = 3 independent experiments). I.P., immunoprecipitation; W.B., Western blot.

Article Snippet: Anti-IRS-1 (06-248), anti-IRS2 (06-506), anti-p85-PI3K (06-195), anti-PTP1B (07-088), anti-phospho-IRS1 (Ser 307 ) (07-247), anti-phospho-IRS1 (Tyr 1179 ) (07-844), catalase (219010), and anti-phosphotyrosine (clone 4G10; 05-321) antibodies were from Merck Millipore (Merck).

Techniques: Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes *

doi: 10.1074/jbc.M113.539189

Figure Lengend Snippet: Effect of PTP1B deficiency in insulin signaling in mice treated chronically with APAP. A, analysis of PTP1B protein content and enzymatic activity in total liver extracts. p85-PI3K (p85) was used as loading control. The results of PTP1B enzymatic activity are expressed as pmol of released phosphate/min/μg protein. *, p < 0.05 APAP-treated PTP1B+/+ versus untreated PTP1B+/+ mice. B, at the end of APAP treatment, the mice were injected with insulin (0.75 unit/kg) or saline, and the hepatic insulin signaling cascade was analyzed by Western blot with the antibodies against phospho-IR (Tyr1162/Tyr1163), IR, phospho-IRS1 (Tyr1179), IRS1, IRS2, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt (n = 4–6 mice per genotype and condition). C, insulin signaling cascade in skeletal muscle was analyzed by Western blot with the antibodies against phospho-IR (Tyr1162/Tyr1163), IR, phospho-Akt (Ser473), phospho-Akt (Thr308), and Akt (n = 4–6 mice per genotype and condition).

Article Snippet: Anti-IRS-1 (06-248), anti-IRS2 (06-506), anti-p85-PI3K (06-195), anti-PTP1B (07-088), anti-phospho-IRS1 (Ser 307 ) (07-247), anti-phospho-IRS1 (Tyr 1179 ) (07-844), catalase (219010), and anti-phosphotyrosine (clone 4G10; 05-321) antibodies were from Merck Millipore (Merck).

Techniques: Activity Assay, Injection, Western Blot