tbg icre  (Vector Biolabs)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: Acute RACK1 deficiency disrupts glucose homeostasis. ( A ) Western blotting analysis of multiple tissues showing liver-specific RACK1 knockout in RACK1 fl/fl mice retro-orbitally injected with AAV8-TBG-GFP (GFP) or AAV8-TBG-iCre (Cre). L, liver; K, kidney; SK, skeletal muscle; H, heart; B, brain. ( B ) Blood glucose levels in GFP- or Cre-injected RACK1 fl/fl mice measured under non-fasted (NF) conditions and after 6 or 18 hours of fasting. ∗ P < .05 compared with GFP controls; 2-tailed Student’s t -test. ( C–F ) Metabolic tolerance tests in GFP and Cre mice: ( C ) insulin tolerance test, ( D ) glucose tolerance test, ( E ) pyruvate tolerance test, and ( F ) glucagon tolerance test. Area under the curve (AUC) quantifications for ( D–F ) are shown below each graph. Group differences over time were analyzed by 2-way repeated-measures ANOVA (time × genotype) with Sidak’s post hoc tests for point-by-point comparisons (n = 6–8 per group). AUCs were compared using 2-tailed unpaired Student’s t -tests. ∗, ∗∗, ∗∗∗ P < .05, .01 and 001 vs GFP, respectively.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Western Blot, Knock-Out, Injection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: RACK1 deficiency impairs hepatic gluconeogenesis in vitro. Primary hepatocytes were isolated from RACK1 fl/fl mice injected with AAV8-TBG-GFP (GFP) or iCre (Cre) and subjected to glucose production assays. ( A ) Glucose output was measured under basal conditions or following stimulation with glucagon (Glu, 200 nM), insulin (Ins, 20 nM), or both. ∗∗, ∗∗∗ P < .01 and .001 vs corresponding basal; # P < .001 vs GFP + Glu; $ P < .05 vs Cre + Glu; & P < .01 vs GFP; 2-way ANOVA followed by Sidak’s post hoc test. ( B ) Cells were treated with vehicle (control) or the PKA inhibitors H89 (5 μM) and compound 3i (0.5 μM) in the absence or presence of glucagon (200 nM). ∗∗∗ P < .001 vs control + basal; & P < .001 vs control + Glu; 2-way ANOVA followed by Sidak’s post hoc test.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: In Vitro, Isolation, Injection, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: RACK1 deficiency attenuates hepatic PKA signaling. ( A–B ) RACK1 fl/fl mice injected with AAV8-TBG-iGFP (GFP) or AAV8-TBG-iCre (Cre) and fasted for 2 hours (basal) or treated with glucagon (100 μg/kg body weight; Glu) or insulin (1.5 U/kg body weight) for 15 minutes. Liver lysates were analyzed for cAMP levels ( A ) and by Western blotting ( B ). Quantified Western blot data are shown in the left panel. ∗∗∗ P < .001 vs corresponding basal. & P < .001 vs GFP + glucagon; 2-way ANOVA followed by Sidak’s post hoc test. ( C ) cAMP levels in primary hepatocytes stimulated with vehicle (basal) or glucagon (200 nM) for 10 minutes. ∗∗∗ P < .001 vs GFP + basal; 2-way ANOVA followed by Sidak’s post hoc test. ( D ) Western blot analysis of primary hepatocytes from GFP and Cre mice stimulated with vehicle (basal) or glucagon (200 nM) for the indicated times. Relative phosphorylation of pCREB S133 and phosphor-PKA substrates (pPKA sub) is expressed as fold change over GFP at 0 minutes, normalized to total protein ( left panel ). ∗, ∗∗∗ P < .05 and .001 vs Glu at 0 minutes, respectively; $ P < .001 vs Cre at 0 minutes; # P < .01 vs corresponding GFP; 2-way ANOVA followed by Sidak’s post hoc test. Arrows indicate pPKA sub unchanged or increased in RACK1-deficient cells. ( E ) Western blot analysis of primary hepatocytes from GFP and Cre mice stimulated with vehicle (basal), or insulin (20 nM) for the indicated times. Relative phosphorylation of pAKT S473 is expressed as fold change over GFP at 0 minutes, normalized to total protein (lower panel). ∗∗∗, # P < .001 vs corresponding Ins at 0 minutes; 2-way ANOVA followed by Sidak’s post hoc test. ( F ) qPCR analysis of PKA target genes G6PC and PCK1 in hepatocytes treated for 4 hours with vehicle, insulin (20 nM), glucagon (100 nM), glucagon + insulin, cAMP (20 μM), or cAMP + insulin. ∗∗∗ P < .001 vs corresponding basal; # P < .001 vs corresponding GFP; % P < .001 vs corresponding cAMP or glucagon; 2-way ANOVA followed by Sidak’s post hoc test. ( G ) qPCR analysis of the indicated genes in liver tissues from GFP and Cre mice following a 6-hour fast. ∗, ∗∗, ∗∗∗ P < .05, .01 and .001 vs GFP, respectively; 2-tailed Student’s t -test.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Injection, Western Blot, Phospho-proteomics

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: Glucagon regulates the dynamic interaction and compartmentation of RACK1 with components of the PKA signaling axis. ( A ) Coimmunoprecipitation of lysates from primary hepatocytes isolated from Alb-Cre/RACK1 fl/fl mice injected with pAd-GFP or pAd-Flag-RACK1 and treated with glucagon (200 nM) for the indicated times. Representative blot showing total proteins in input lysates and immunoprecipitated proteins in pellet fractions. ( B ) Quantification of RACK1-associated proteins from ( A ) after subtraction of GFP background, shown as fold enrichment relative to 0 minutes. ∗∗, ∗∗∗ P < .01 and .001 vs 0 minutes, respectively; n = 3; 1-way ANOVA followed by Dunnett’s post hoc test. ( C–D ) Western blot analysis of plasma membrane ( C ) and nuclear ( D ) fractions from primary hepatocytes isolated from RACK1 fl/fl mice injected with AAV8-TBG-iGFP (GFP) or AAV8-TBG-iCre (Cre) and stimulated with glucagon (200 nM) for the indicated time. Glucagon-induced changes in protein localization were quantified as fold change relative to 0 minutes in GFP cells and values are indicated below each blot. Quantitative data from multiple experiments are presented in left panels. ∗, ∗∗, ∗∗∗ P < .05, .01 and .001 vs GFP at 0 minutes, respectively; # P < .001 vs corresponding GFP; 2-way ANOVA followed by Sidak’s post hoc test.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Isolation, Injection, Immunoprecipitation, Western Blot, Clinical Proteomics, Membrane

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A Dual-compartment Scaffolding Role for Receptor for Activate C Kinase 1 in Hepatic Glucagon Signaling and Gluconeogenesis

doi: 10.1016/j.jcmgh.2025.101666

Figure Lengend Snippet: Rescue of glucose homeostasis defects induced by acute RACK1 deficiency via expression of constitutively active PKAcα W196R . ( A ) qPCR analysis of PKA target gene expression in livers of RACK1 fl/fl mice injected with AAV-TBG-GFP (RACK1 fl/fl /GFP), RACK1 fl/fl mice injected with AAV-TBG-Cre (RACK1 fl/fl /Cre), PKAca W196R mice injected with AAV-TBG-Cre (PKACA/Cre), or RACK1 fl/fl /PKAca W196R mice injected with AAV-TBG-Cre (RACK1 fl/fl /PKACA/Cre). ∗, ∗∗∗ P < .05 and .001 vs corresponding RACK1 fl/fl /GFP, respectively; 1-way ANOVA followed by Dunnett’s post hoc test. ( B ) Blood glucose levels in the indicated mice following an 18-hour fast. ∗, ∗∗ P < .05 and .01 vs RACK1 fl/fl /GFP, respectively; 1-way ANOVA followed by Dunnett’s post hoc test (n = 8). ( C–F ) Glucose tolerance test ( C ) with corresponding area under the curve (AUC) analysis ( D ), pyruvate tolerance test ( E ) with corresponding AUC analysis ( F ). Group differences over time were analyzed by 2-way repeated-measures ANOVA (time × genotype) with Sidak’s post hoc tests for point-by-point comparisons (n = 5–8 per group). AUCs were compared using 1-way ANOVA followed by Dunnett’s post hoc test. ∗, ∗∗, ∗∗∗ P < .05, .01, and .001 vs RACK1 fl/fl /GFP, respectively.

Article Snippet: Acute deletion of RACK1 gene in the liver was achieved through the injection of AAV viruses (1 × 10 11 genome copies/mouse) encoding Cre recombinase under the TBG promoter (AAV-TBG-iCre; Vector Biolabs) into the retroorbital vein of RACK1 fl/fl transgenic mice.

Techniques: Expressing, Targeted Gene Expression, Injection