anti phosphorylated p insulin receptor substrate 1  (Cell Signaling Technology Inc)

Journal: Experimental and Therapeutic Medicine

Article Title: Sodium sulphate ameliorates hypercholesterolemia via the upregulation of Cyp7a1 in hepatocytes and alleviates hepatic insulin resistance via the downregulation of Trib3 in mice with high cholesterol diets

doi: 10.3892/etm.2024.12650

Figure Lengend Snippet: Effect of sodium sulphate on the insulin signaling pathway in the liver tissues of mice fed an HCD. (A) Kyoto Encyclopedia of Genes and Genomes analysis of the biological pathways of differently expressed mRNAs in the livers of mice from the CON vs. HCD and HCD vs. HCD + MSS groups. (B) RT-qPCR and western blotting results of the Trib3 mRNA and TRB3 protein expression levels in the liver tissues of mice from the CON, HCD, HCD + LSS, HCD + MSS and HCD + HSS groups, respectively. (C) Western blotting results showing the protein expression levels of p-IRS1 (Tyr608), IRS1, p-AKT and AKT in the liver tissues of mice from the CON, HCD, HCD + LSS, HCD + MSS and HCD + HSS groups. The right panel shows the semi-quantitative analysis of the p-AKT/AKT ratio. GAPDH was used as the internal control for western blotting and RT-qPCR. ## P<0.01, ### P<0.001 vs. the CON group; * P<0.05, ** P<0.01, *** P<0.001 vs. the HCD group. HCD, high cholesterol diet; CON, control; LSS, low dose of sodium sulphate; MSS, middle dose of sodium sulphate; HSS, high dose of sodium sulphate; RT-qPCR, reverse transcription-quantitative PCR; Trib3/TRB3, tribbles pseudokinase 3; p-, phosphorylated; IRS1, insulin receptor substrate 1.

Article Snippet: The primary antibodies comprised: Rabbit anti-GAPDH (1:1,000; cat. no. 2118S; CST Biological Reagents Co., Ltd.), mouse anti-CYP7A1 (1:1,000; cat. no. 2683295; MilliporeSigma), rabbit anti-hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 7 (HSD3B7; 1:1,000; cat. no. ab190223; Abcam), rabbit anti-aldo-keto reductase family 1 member D1 (AKR1D1; 1:1,000; cat. no. ab101393; Abcam), rabbit anti-acyl-CoA oxidase 2 (1:1,000; cat. no. ab197808; Abcam), rabbit anti-hydroxysteroid 17-β dehydrogenase 4 (HSD17B4; 1:1,000; cat. no. ab97971; Abcam), rabbit anti-CYP27A1 (1:2,000; cat. no. ab126785; Abcam), rabbit anti-CYP39A1 (1:1,000; cat. no. ab129334; Abcam), rabbit anti-isopentenyl-diphosphate δ isomerase 1 (IDI1; 1:3,000; cat. no. ab97448; Abcam), rabbit anti-lanosterol synthase [anti-oxidosqualene-lanosterol cyclase (OSC); 1:1,000; cat. no. ab80364; Abcam], rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1; 1:1,000; cat. no. ab195046; Abcam), rabbit anti-farnesyl diphosphate synthase (FDPS; 1:2,000; cat. no. ab153805; Abcam), rabbit anti-mevalonate diphosphate decarboxylase (MVD; 1:1,000; cat. no. ab96226; Abcam), rabbit anti-mevalonate kinase (MVK; 1:1,000; cat. no. ab154515; Abcam), rabbit anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR; 1:1,000; cat. no. ab174830; Abcam), rabbit anti-low density lipoprotein receptor (LDLR; 1:1,000; cat. no. ab52818; Abcam), rabbit anti-c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252s; CST Biological Reagents Co., Ltd.), mouse anti-phosphorylated (p)-JNK (1:2,000; cat. no. 9255s; CST Biological Reagents Co., Ltd.), rabbit anti-p-insulin receptor substrate 1 (p-IRS1; Tyr608) mouse (1:1,000; cat. no. 09-432; MilliporeSigma), rabbit anti-IRS-1 (1:1,000; cat. no. 2382S; CST Biological Reagents Co., Ltd.), rabbit anti-p-AKT (Ser473) (1:1,000; cat. no. 4060S; CST Biological Reagents Co., Ltd.), rabbit anti-AKT (1:1,000; cat. no. 4685S; CST Biological Reagents Co., Ltd.), mouse anti-tribbles pseudokinase 3 (TRB3; 1:100; cat. no. sc-390242; Santa Cruz Biotechnology, Inc.), rabbit anti-c-Jun (1:1,000; cat. no. ab32137; Abcam), mouse anti-p-c-Jun (1:200; cat. no. sc-822; Santa Cruz Biotechnology, Inc.) and goat anti-KLB (0.5 µg/ml; cat. no. AF5889; R&D Systems China Co., Ltd.).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: Sodium sulphate ameliorates hypercholesterolemia via the upregulation of Cyp7a1 in hepatocytes and alleviates hepatic insulin resistance via the downregulation of Trib3 in mice with high cholesterol diets

doi: 10.3892/etm.2024.12650

Figure Lengend Snippet: Sodium sulphate ameliorates hypercholesterolemia and hepatic insulin resistance in mice fed an HCD. Sodium sulphate inhibits the highly expressed TRB3 in the hepatocytes of mice fed an HCD, to attenuate the hepatic insulin resistance of these mice. In the enterocytes of the ileum, FGF15 expression is downregulated following the administration of sodium sulphate to mice fed an HCD. In addition, the hepatic expression of KLB, which is the co-receptor of FGFR4 for FGF15 binding, is significantly downregulated by sodium sulphate, and reduces the activation of JNK, which is downstream of the FGF15/FGFR4-KLB signaling pathway. The activation of c-Jun, which is the major target of p-JNK, is notably reduced, and the expression of Cyp7a1 , which is inhibited by p-c-Jun, is significantly increased, which enhances the conversion of cholesterols to bile acids in these mice. HCD, high cholesterol diet; TRB3, tribbles homolog 3; FGF, fibroblast growth factor; FGFR, FGF receptor; KLB, Klotho β; JNK, c-Jun N-terminal kinase; p-, phosphorylated; IRS1, insulin receptor substrate 1; PDK1, 3-phosphoinositide-dependent kinase 1; FXR, farnesoid X receptor.

Article Snippet: The primary antibodies comprised: Rabbit anti-GAPDH (1:1,000; cat. no. 2118S; CST Biological Reagents Co., Ltd.), mouse anti-CYP7A1 (1:1,000; cat. no. 2683295; MilliporeSigma), rabbit anti-hydroxy-δ-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 7 (HSD3B7; 1:1,000; cat. no. ab190223; Abcam), rabbit anti-aldo-keto reductase family 1 member D1 (AKR1D1; 1:1,000; cat. no. ab101393; Abcam), rabbit anti-acyl-CoA oxidase 2 (1:1,000; cat. no. ab197808; Abcam), rabbit anti-hydroxysteroid 17-β dehydrogenase 4 (HSD17B4; 1:1,000; cat. no. ab97971; Abcam), rabbit anti-CYP27A1 (1:2,000; cat. no. ab126785; Abcam), rabbit anti-CYP39A1 (1:1,000; cat. no. ab129334; Abcam), rabbit anti-isopentenyl-diphosphate δ isomerase 1 (IDI1; 1:3,000; cat. no. ab97448; Abcam), rabbit anti-lanosterol synthase [anti-oxidosqualene-lanosterol cyclase (OSC); 1:1,000; cat. no. ab80364; Abcam], rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1; 1:1,000; cat. no. ab195046; Abcam), rabbit anti-farnesyl diphosphate synthase (FDPS; 1:2,000; cat. no. ab153805; Abcam), rabbit anti-mevalonate diphosphate decarboxylase (MVD; 1:1,000; cat. no. ab96226; Abcam), rabbit anti-mevalonate kinase (MVK; 1:1,000; cat. no. ab154515; Abcam), rabbit anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR; 1:1,000; cat. no. ab174830; Abcam), rabbit anti-low density lipoprotein receptor (LDLR; 1:1,000; cat. no. ab52818; Abcam), rabbit anti-c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252s; CST Biological Reagents Co., Ltd.), mouse anti-phosphorylated (p)-JNK (1:2,000; cat. no. 9255s; CST Biological Reagents Co., Ltd.), rabbit anti-p-insulin receptor substrate 1 (p-IRS1; Tyr608) mouse (1:1,000; cat. no. 09-432; MilliporeSigma), rabbit anti-IRS-1 (1:1,000; cat. no. 2382S; CST Biological Reagents Co., Ltd.), rabbit anti-p-AKT (Ser473) (1:1,000; cat. no. 4060S; CST Biological Reagents Co., Ltd.), rabbit anti-AKT (1:1,000; cat. no. 4685S; CST Biological Reagents Co., Ltd.), mouse anti-tribbles pseudokinase 3 (TRB3; 1:100; cat. no. sc-390242; Santa Cruz Biotechnology, Inc.), rabbit anti-c-Jun (1:1,000; cat. no. ab32137; Abcam), mouse anti-p-c-Jun (1:200; cat. no. sc-822; Santa Cruz Biotechnology, Inc.) and goat anti-KLB (0.5 µg/ml; cat. no. AF5889; R&D Systems China Co., Ltd.).

Techniques: Expressing, Binding Assay, Activation Assay

Journal: Diabetes & Metabolism Journal

Article Title: Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy

doi: 10.4093/dmj.2022.0332

Figure Lengend Snippet: Extracellular vimentin-induced glucose transporter type 1 (GLUT1) expression depends on insulin-like growth factor 1 receptor (IGF1R) and activation of extracellular-signal-regulated kinase (ERK). (A) Western blot analyses for phospho-insulin receptor substrate 1 (P-IRS1; Tyrosine 612), P-Akt (Serine 473), and P-IGF1R (Tyrosine 1158+1162+1163) were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Samples from three different batches of treatment were loaded onto a gel ( n =3). (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for GLUT1, GLUT4, hypoxia-inducible factor 1α (Hif-1α) were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with anti-IGF1R antibody (11 µg/mL) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (GLUT1, n =3; GLUT4, n =3; Hif-1α, n =4). (C) Western blotting for P-ERK1/2 (Threonine 202/Tyrosine 204) was performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using GAPDH as an internal control ( n =3). (D) qRT-PCR analyses for GLUT1, GLUT4, Hif-1α were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with U0126 (ERK inhibitor, 25 µM) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control ( n =3 for each). a P <0.05, b P <0.01, c P <0.001.

Article Snippet: Antibodies for GLUT1, GLUT4, phospho-insulin receptor substrate 1 (P-IRS1), P-Akt, phospho-extracellular-signal-regulated kinase (P-ERK), phospho-hormone-sensitive lipase (P-HSL; S563), HSL, peroxisome proliferator-activated receptor γ (PPARγ), P-ERK, inositol-requiring enzyme 1 α (IRE1α), C/EBP homologous protein (CHOP), p62, and microtubule-associated proteins 1A/1B light chain 3 (LC3)-I/II were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay, Western Blot, Derivative Assay, Recombinant, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Incubation

Journal: Diabetes & Metabolism Journal

Article Title: Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy

doi: 10.4093/dmj.2022.0332

Figure Lengend Snippet: Extracellular vimentin promotes glucose uptake and free fatty acid (FFA) uptake via changes in expression of glucose transporters and fatty acid transporter. (A) 3T3-L1-derived adipocytes were treated with recombinant vimentin (20 µg/mL for 24 hours), insulin (1 µM for 30 minutes) or both vimentin (20 µg/mL for 24 hours) and insulin (1 µM for 30 minutes). 2-Deoxyglucose (2-DG) uptake was measured ( n =4). (B) 3T3-L1-derived adipocytes were treated with or without recombinant vimentin (20 µg/mL) for 24 hours and FFA uptake was measured using fluorometric fatty acid dye-loading solution (TF2-C12 Fatty Acid) uptake assay ( n =3). (C) Western blot analyses for CD36, glucose transporter type 4 (GLUT4), and GLUT1 were performed using fractionated lysates (plasma membrane and cytosol) of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using either caveolin-1 (BD Biosciences) as a plasma membrane marker, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytosol marker ( n =3). (D) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for CD36, GLUT1, and GLUT4 were performed using RNA from 3T3-L1-derived adipocytes cultured with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control ( n =5, n =6, n =4). (E) Western blot analyses for GLUT1 and GLUT4 using total lysates of 3T-3L1-derived adipocytes treated with or with recombinant vimentin (20 µg/mL) for 24 hours. Band quantification was performed using GAPDH, as an internal control ( n =3). (F) Western blot analyses for GLUT1 and GLUT4 were performed using total lysates of 3T3-L1-derived adipocytes with or with recombinant vimentin (20 µg/mL) for 36 hours. Band quantification was performed using α-tubulin (AbFrontier), an internal control ( n =3). (G) qRT-PCR analysis for hypoxia-inducible factor 1α (Hif-1α) was performed using RNA from 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control ( n =4). RFU, relative fluorescence unit. a P <0.05, b P <0.01, c P <0.001.

Article Snippet: Antibodies for GLUT1, GLUT4, phospho-insulin receptor substrate 1 (P-IRS1), P-Akt, phospho-extracellular-signal-regulated kinase (P-ERK), phospho-hormone-sensitive lipase (P-HSL; S563), HSL, peroxisome proliferator-activated receptor γ (PPARγ), P-ERK, inositol-requiring enzyme 1 α (IRE1α), C/EBP homologous protein (CHOP), p62, and microtubule-associated proteins 1A/1B light chain 3 (LC3)-I/II were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Derivative Assay, Recombinant, Western Blot, Clinical Proteomics, Membrane, Marker, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Cell Culture, Control, Fluorescence

Journal: Diabetes & Metabolism Journal

Article Title: Extracellular Vimentin Alters Energy Metabolism And Induces Adipocyte Hypertrophy

doi: 10.4093/dmj.2022.0332

Figure Lengend Snippet: Extracellular vimentin-induced glucose transporter type 1 (GLUT1) expression depends on insulin-like growth factor 1 receptor (IGF1R) and activation of extracellular-signal-regulated kinase (ERK). (A) Western blot analyses for phospho-insulin receptor substrate 1 (P-IRS1; Tyrosine 612), P-Akt (Serine 473), and P-IGF1R (Tyrosine 1158+1162+1163) were performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Samples from three different batches of treatment were loaded onto a gel ( n =3). (B) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses for GLUT1, GLUT4, hypoxia-inducible factor 1α (Hif-1α) were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with anti-IGF1R antibody (11 µg/mL) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control (GLUT1, n =3; GLUT4, n =3; Hif-1α, n =4). (C) Western blotting for P-ERK1/2 (Threonine 202/Tyrosine 204) was performed using lysates of 3T3-L1-derived adipocytes treated with or without recombinant vimentin (20 µg/mL) for 30 minutes. Band quantification was performed using GAPDH as an internal control ( n =3). (D) qRT-PCR analyses for GLUT1, GLUT4, Hif-1α were performed with RNA from 3T3-L1-derived adipocytes treated or untreated with U0126 (ERK inhibitor, 25 µM) for 1 hour and then incubated with or without recombinant vimentin (20 µg/mL) for 24 hours. GAPDH was used as an internal control ( n =3 for each). a P <0.05, b P <0.01, c P <0.001.

Article Snippet: Antibodies for GLUT1, GLUT4, phospho-insulin receptor substrate 1 (P-IRS1), P-Akt, phospho-extracellular-signal-regulated kinase (P-ERK), phospho-hormone-sensitive lipase (P-HSL; S563), HSL, peroxisome proliferator-activated receptor γ (PPARγ), P-ERK, inositol-requiring enzyme 1 α (IRE1α), C/EBP homologous protein (CHOP), p62, and microtubule-associated proteins 1A/1B light chain 3 (LC3)-I/II were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Activation Assay, Western Blot, Derivative Assay, Recombinant, Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Incubation

Journal: Cells

Article Title: Psoralen Suppresses Lipid Deposition by Alleviating Insulin Resistance and Promoting Autophagy in Oleate-Induced L02 Cells

doi: 10.3390/cells11071067

Figure Lengend Snippet: Psoralen alleviates insulin resistance in sodium oleate-induced L02 cells by enhancing the expression of GLUT4 and promoting the GLUT4 membrane translocation. L02 cells except the control group were induced with sodium oleate (100 μM) for 24 h to induce lipid deposition. Then, all cells were stimulated by insulin (10 μg/mL) for 30 min and then incubated with psoralen at 0, 0.37, 1.1, and 3.3 μM for 24 h. ( A ) The glucose content was measured by the glucose content assay kit. ( B ) The expression of GLUT4 and GLUT2 was detected by Western blotting. ( C ) The expression of INSR, p-INSR, IRS1, and p-IRS1 were measured by Western blotting. ( D ) The expression of GLUT4 and F-actin was detected by immunofluorescence and images captured by confocal microscopy (scale bar = 10 μm). ( E ) Colocalization efficiency of GLUT4 and F-actin in multiple cells ( n ≥ 3 cells) within the field of view was quantified by line scan analysis (160 pixels with two ends on the membrane) by observing the overlap of fluorescence intensity peaks along the spanning contour. ( F ) The expression of GLUT2 and F-actin was detected by immunofluorescence and images captured by confocal microscopy (scale bar = 10 μm). ( G ) Colocalization efficiency of GLUT2 and F-actin in multiple cells ( n ≥ 3 cells) within the field of view was quantified by line scan analysis (250 pixels with two ends on the membrane) by observing the overlap of fluorescence intensity peaks along spanning profiles. All values were expressed as the mean ± SD from three independent experiments. # p < 0.05, ## p < 0.01, vs. the control group; ** p < 0.01 vs. sodium oleate-induced group. Abbreviations: INS, insulin; Pso, psoralen; INSR, insulin receptor; IRS1, insulin receptor substrate 1.

Article Snippet: AMPK, phosphorylated AMPK (p-AMPK), acetyl-CoA carboxylase (ACC), phosphorylated acetyl-CoA carboxylase (p-ACC), insulin receptor (INSR), phosphorylated insulin receptor (p-INSR), insulin receptor substrate 1 (IRS1), and phosphorylated insulin receptor substrate 1 (p-IRS1) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Translocation Assay, Incubation, Western Blot, Immunofluorescence, Confocal Microscopy, Fluorescence