rat mouse glut4  (Jackson Immuno)

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a , b Osteogenic activity of MC3T3-E1 osteoblastic cells with 11β-HSD1 overexpression (MC3T3-HSD1 cells) and transfected control cells (MC3T3-GFP cells). a Alkaline phosphatase (ALP) and alizarin red staining. b The mRNA expression of RUNX family transcription factor 2 ( Runx2 ) and bone gamma-carboxyglutamate protein ( Bglap ). c , d Combination analysis of RNA sequencing (RNA-seq) and assays for transposase-accessible chromatin sequencing (ATAC-seq) in MC3T3-GFP cells and MC3T3-HSD1 cells. c Intersection of differential genes from RNA-seq and ATAC-seq. d Biological processes associated with Egr2. e The mRNA and protein expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells. f The mRNA expression of Egr2 during osteogenic differentiation. g , h The osteogenic activity of MC3T3-HSD1 cells with Egr2 overexpressing. g Alizarin red staining. h Bglap mRNA expression. i The glucose uptake test of MC3T3-GFP cells and MC3T3-HSD1 cells. j , k The mRNA expression of key glucose transporter proteins and components of insulin-dependent glucose uptake pathway in MC3T3-GFP cells and MC3T3-HSD1 cells. j Glucose transporter type 1 (Glut1) , 3 (Glut3) and 4 (Glut4) . k: Insulin receptor (Ir), Insulin receptor substrate 1 (Irs1), Phosphatidylinositol-3-kinase catalytic subunit regulatory subunit 1 (Pik3r), alpha (Pik3ca) and subunit beta (Pik3cb) . l , m The glucose uptake test and mRNA expression of Glut4 and Pik3cb in MC3T3-HSD1 cells with Egr2 overexpressing. l Glucose uptake test. m Glut4 and Pik3cb . n Dual-luciferase reporter analysis and ChIP quantitative polymerase chain reaction (ChIP-qPCR) analysis of Ege2 target interactions for Pik3cb and Glut4 gene promotors. n Pik3cb and Glut4 gene promotors. o ChIP-qPCR analysis. p Pik3cb and Glut4 gene promotors with different mutations. Note: Data were presented as mean value ± SEM for ( a , b , e – m , p ). n = 3 biologically independent samples for RNA-seq, ATAC-seq, western blot analysis, glucose uptake test and osteogenic staining; n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using two-tailed Student’s t-test ( e ), one-way ANOVA followed by Tukey’s post-hoc test ( n , p ), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli ( a , b , f – m ) to adjust for multiple comparisons. All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Activity Assay, Over Expression, Transfection, Control, Staining, Expressing, RNA Sequencing Assay, Sequencing, Luciferase, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a The mRNA expression of glucocorticoid target gene Glucocorticoid-induced leucine zipper ( Gilz ) in MC3T3-GFP cells and MC3T3-HSD1 cells after treating with 11β-HSD1 inhibitor (AZD8329). b , c The mRNA and protein expression of early growth response 2 ( Egr2 ) in MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. b The mRNA expression. c The protein expression. d The mRNA expression of Egr2 in MC3T3-GFP cells and MC3T3-HSD1 cells after treating with AZD8329 during osteogenic differentiation. e – h The osteogenic activity of MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. e Alizarin red staining. f Bone gamma-carboxyglutamate protein (Bglap) mRNA expression. g Runt-related transcription factor 2 (Runx2) mRNA expression. h Osterix mRNA expression. i – j The glucose uptake test and mRNA expression levels of Glucose transporter type 4 (Glut4) and Phosphatidylinositol-3-kinase catalytic subunit beta (Pik3cb) in MC3T3-GFP cells and MC3T3-HSD1 cells after treatment of AZD8329. g: Glucose uptake test. h Glut4 and Pik3cb mRNA expression. Note: Data were presented as mean value ± SEM for ( a , b , d – j ). n = 3 biologically independent samples for osteogenic staining, western blot analysis and LC-MS/MS analysis, n = 6 biologically independent samples for RT-qPCR analysis. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test ( a – c ), and two-way ANOVA followed by a two-stage step-up method by Benjamini, Krieger and Yekutieli ( d – j ) to adjust for multiple comparisons. All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Expressing, Activity Assay, Staining, Western Blot, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Targeting osteoblastic 11β-HSD1 to combat high-fat diet-induced bone loss and obesity

doi: 10.1038/s41467-024-52965-4

Figure Lengend Snippet: a Design of the bone-targeted 11β-HSD1 inhibitor, (DSS) 6 -AZD8329. b , c Organ distribution and skeletal distribution of (DSS) 6 -AZD8329 ( n = 3) and (RKK) 6 -AZD8329 ( n = 3). b The organ distribution. c The skeletal distribution, scale bar=20 μm. d Experimental design of wild-type mice treated with (DSS) 6 -AZD8329 and (RKK) 6 -AZD8329. e – g The micro-CT analysis and bone histometric analysis. e The trabecular bone and cortical bone microstructure. f The percentage change of trabecular bone volume/total volume (Tb. BV/TV) and trabecular bone density (Tb. v. BMD). g The percentage change of cortical bone density (Ct. v. BMD). h The calcein double labeling, scale bar=20 μm. i The percentage change of mineral apposition rate (MAR), bone formation rate per bone surface (BFR/BS) and the number of osteoblasts per bone surface (N.Ob/BS). j – k Weight gain and energy intake during HFD. * P < 0.05, ** P < 0.01, *** P < 0.001 when other groups vs. HFD+vehicle group. j Weight gain. k Energy intake. l Weights and representative photographs of gonadal white adipose tissues (gWAT). m – o Glucose handling tests ( n = 8 for two vehicle-treated groups, n = 12 for two drug-treated groups). * P < 0.05, ** P < 0.01, *** P < 0.001 when other groups vs. HFD + vehicle group. m Fasting blood glucose. n: Insulin tolerance test (ITT). o Oral glucose tolerance test (oGTT). p Glucose uptake tests ( n = 8 for two vehicle-treated groups, n = 12 for two drug-treated groups). Left: Representative images of glucose uptake. Right: Quantitative analysis of glucose uptake into liver, WAT, muscle and bone. q Skeletal mRNA expression of Hsd11b1 , Gilz , Egr2 , Pik3cb and Glut4 . Note: Data were presented as mean value ± SEM for ( f , g , i – q ). Chow+vehicle ( n = 8), mice with Chow feeding and start administration of vehicle since week 8; HFD+vehicle ( n = 8), HFD + (RKK) 6 -AZD8329 ( n = 13), HFD + (DSS) 6 -AZD8329 ( n = 13), mice with HFD feeding and start administration of vehicle, (RKK) 6 -AZD8329, or (DSS) 6 -AZD8329, respectively, since week 8. All samples are biologically independent samples. Statistical significance was calculated using one-way ANOVA followed by Tukey’s post-hoc test ( f – g , i , l , m – q ) and two-way ANOVA followed by Sidak’s multiple comparisons test ( j – k , n – o ). All tests were two-sided.

Article Snippet: Following blocking with 5% nonfat milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies (1:500) against Actin (4967, Cell Signaling, USA), p110β (ab151549, abcam, UK), AKT (4691 T, Cell Signaling, USA), phosphorylated AKT (4060 T, pAKT, Cell Signaling, USA), Glut4 (21619, SAB, USA), 11β-HSD1 (AF3397, R&D system, USA), Egr2 (EPR4004, Abcam, UK), H6PDH (EPR12338, Abcam, UK).

Techniques: Micro-CT, Labeling, Expressing

Journal: Heliyon

Article Title: Effects of Salvia mirzayanii extract administration on hyperglycemia improvement in diabetic rats: The role of GLUT4, PEPCK and G6Pase genes

doi: 10.1016/j.heliyon.2024.e25256

Figure Lengend Snippet: Primer sequences and expected ampliocn size of the genes for RT-qPCR analysis.

Article Snippet: Slides were then treated with mouse anti-rat GLUT4 mAb (Cell Signaling Technology, USA), anti-rat G6Pase polyclonal antibody (Biorbyt Ltd, United Kingdom) and mouse anti-rat PEPCK mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 10 μg/mL concentration.

Techniques: Sequencing

Journal: Heliyon

Article Title: Effects of Salvia mirzayanii extract administration on hyperglycemia improvement in diabetic rats: The role of GLUT4, PEPCK and G6Pase genes

doi: 10.1016/j.heliyon.2024.e25256

Figure Lengend Snippet: Analysis of GLUT4 expression in the muscle tissue in normal control (NC), diabetic control (DC) and diabetic control treated with S. mirzayanii (DC-Treated with SM). (A) Analysis of mRNA expression using RT-qPCR. Data were normalized with that of β-actin and then calculated as relative to the NC group. Each value represents the mean ± SEM (n = 10 per group) in two independent experiments. P < 0.01: Significant difference vs. control group. (B) Protein expression level analysis by IHC technique in NC, DC and DC-Treated with SM. The small white arrows indicate the expression of GLUT4 in the muscle tissue.

Article Snippet: Slides were then treated with mouse anti-rat GLUT4 mAb (Cell Signaling Technology, USA), anti-rat G6Pase polyclonal antibody (Biorbyt Ltd, United Kingdom) and mouse anti-rat PEPCK mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 10 μg/mL concentration.

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

doi: 10.1152/ajpendo.00020.2016

Figure Lengend Snippet: Fig. 7. Impact of muscle CPT1mt expression on muscle glucose metabolism in dbTg-HFHS mice. WT and dbTg mice fed Ctrl or HFHS diet for 16 wk were euthanized in the postprandial state after a 2-h fast. The gastrocnemius protein expression level of Glut4, phosphorylated (pS293PDH), and total pyruvate dehydrogenase (PDH), PDH kinases 1 and 4 (PDK1, PDK4) (A) as well as phosphorylated (pS641GS) and total glycogen synthase (GS; B) was analyzed by Western blotting using GAPDH as loading control. The same membranes as in Fig. 6A were used for Fig. 7B. Quantification and determination of the p-PDH/PDH and p-GS/GS ratios relative to WT-Ctrl mice are shown (n 5–6/group). C: gastrocnemius glycogen content in WT-Ctrl (n 9), WT-HFHS (n 9), and dbTg-HFHS (n 11) mice. Results are means SE. $P 0.05, $$P 0.01, $$$P 0.001, dbTg-HFHS vs. WT-Ctrl mice; *P 0.05, **P 0.01, dbTg-HFHS vs. WT-HFHS mice.

Article Snippet: Primary antibodies were directed against mouse total and phosphorylated (Ser473) Akt (cs-9272 and cs-9271, Cell Signaling Technology), rat CPT1A (30), rat CPT1B (a gift from VA Zammit, Warwick University, Warwick, UK), human CPT2 (4), human total and phosphorylated (Tyr202/204) ERK (cs-9102 and cs-9101, Cell Signaling Technology), mouse GAPDH (sc-25778; Santa Cruz Biotechnology), rabbit total and phosphorylated (Ser641) glycogen synthase (GS) (cs-3886 and cs-3891; Cell Signaling Technology), human total and phosphorylated (Ser9) GSK-3 (cs-9315 and cs-9336, Cell Signaling Technology), rat Glut4 (cs-2213, Cell Signaling Technology), human total PDH E1 subunit (MSP03-SP, MitoSciences), human phosphorylated (Ser293) PDH E1 subunit (ab110330, Abcam), human PDK1 (ab110025, Abcam), human PDK4 (ab71240, Abcam), and sea urchin -tubulin (Tyr5168, Sigma).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: The Effect of Gonadotropin on Glucose Transport and Apoptosis in Rat Ovary

doi: 10.1371/journal.pone.0042406

Figure Lengend Snippet: Ovaries from immature rats were fixed and adjacent paraffin sections were cut. Glut1, Glut2, Glut3 and Glut4 proteins were localized with corresponding specific antibodies by ABC (avidin -biotin complex) method. Column Glut show positivities and immunoreactivities for Glut1, Glut2, Glut3 and Glut4 respectively by IHC. Note that Glut1–4 were not only detected in granulosa cells, theca cells but also in oocyte. Each independent data came form six ovaries. Magnification, 400×, Bar = 20 µm.

Article Snippet: Mouse monoclonal anti-rat Glut4 was from Cell Signaling (Cell Signaling, USA).

Techniques: Avidin-Biotin Assay

Journal: PLoS ONE

Article Title: The Effect of Gonadotropin on Glucose Transport and Apoptosis in Rat Ovary

doi: 10.1371/journal.pone.0042406

Figure Lengend Snippet: The ovaries were also collected for Glut mRNA analysis by real-time PCR and RT-PCR. The Glut mRNA abundance were normalized by 18S rRNA and β-actin. Although eCG significantly increased Glut1, Glut3 and Glut4 mRNA level, these responses were abrogated by eCG antiserum. Neither eCG alone nor the combination of eCG and eCG antiserum elicited a significant response in the Glut2 mRNA regulation. **, P<0.01.

Article Snippet: Mouse monoclonal anti-rat Glut4 was from Cell Signaling (Cell Signaling, USA).

Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction