Journal: Ecotoxicology and environmental safety

Article Title: MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

doi: 10.1016/j.ecoenv.2021.112130

Figure Lengend Snippet: Fig. 2. Chronic exposure to arsenite increases miR-191 levels, blocks the IRS1/AKT pathway, and decreases membrane protein levels of GLUT4 in the livers of mice. Mice were exposed to arsenite (0 or 20 ppm) in drinking water for 12 months. (A) The levels of miR-191 in livers were determined by qRT-PCR assays (mean ± SD, n = 6). (B) Western blots of total protein were performed, and relative protein levels of p-IRS1, IRS1, and p-AKT in livers were measured (mean ± SD, n = 4). (C) Western blots of membrane and cytoplasm protein were performed, and relative protein levels of GLUT4 in livers were measured (mean ± SD, n = 3). #

Article Snippet: The levels of GLUT4 were assessed by Western blots with anti-GLUT4 antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Membrane, Quantitative RT-PCR, Western Blot

Journal: Ecotoxicology and environmental safety

Article Title: MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

doi: 10.1016/j.ecoenv.2021.112130

Figure Lengend Snippet: Fig. 3. Arsenite induces increases of miR-191 levels, inhibition of the IRS1/AKT pathway and translocation of GLUT4 in insulin-treated L-02 cells. L-02 cells were treated with 0, 1, 2, 4, 6, 8, 10, 20, or 30 μM arsenite for 24 h and then incubated with insulin (100 nM) for 30 min (A) Cell viability was determined by CCK-8 assays (mean ± SD, n = 6). L-02 cells were exposed to arsenite (0, 1, 2, 4, or 8 µM) for 24 h and then incubated with insulin (100 nM) for 30 min (B) Glucose consumption and glycogen levels in L-02 cells were measured by glucose assay kits and glycogen assay kits, respectively (mean ± SD, n = 3). (C) The levels of miR- 191 in L-02 cells were determined by qRT-PCR assays (mean ± SD, n = 3). (D) Western blots of total protein were performed, and relative protein levels of p-IRS1, IRS1, and p-AKT in L-02 cells were measured (mean ± SD, n = 3). (E) Western blots of membrane and cytoplasm protein were performed, and relative protein levels of GLUT4 in L-02 cells were measured (mean ± SD, n = 3). (F) Immunofluorescence staining of GLUT4 and WGA in L-02 cells for the indicated groups were per formed. Blue represents nuclear DNA staining by DAPI; green represents WGA staining; red represents GLUT4 staining. Bar = 50 µm. #p < 0.05, different from L-02 cells in the absence of arsenite. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The levels of GLUT4 were assessed by Western blots with anti-GLUT4 antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Translocation Assay, Incubation, CCK-8 Assay, Glucose Assay, Quantitative RT-PCR, Western Blot, Membrane, Immunofluorescence, Staining

Journal: Ecotoxicology and environmental safety

Article Title: MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

doi: 10.1016/j.ecoenv.2021.112130

Figure Lengend Snippet: Fig. 4. The AKT pathway is involved in the arsenite-induced decreases of glucose consumption and glycogen accumulation, and the translocation of GLUT4 in insulin-treated L-02 cells. L-02 cells were exposed to 0 or 4 μM arsenite for 24 h, then exposed to 0 or 10 μg/ml SC79 (agonist of AKT) for 30 min, after which they were treated with insulin (100 nM) for 30 min (A) Western blots of total protein were performed, and relative protein levels of p-AKT in L-02 cells were measured (mean ± SD, n = 3). (B) Glucose consumption and glycogen levels in L-02 cells were measured by glucose assay kits and glycogen assay kits, respectively (mean ± SD, n = 3). (C) Western blots of membrane and cytoplasm protein were performed, and relative protein levels of GLUT4 in L-02 cells were measured (mean ± SD, n = 3). (D) Immunofluorescence staining of GLUT4 and WGA in L-02 cells were performed for the indicated groups. Blue represents nuclear DNA staining by DAPI; green represents WGA staining; red represents GLUT4 staining. Bar = 50 µm. # p < 0.05, different from L-02 cells in the absence of arsenite; *p < 0.05, different from arsenite-exposed L-02 cells in the absence of SC79. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The levels of GLUT4 were assessed by Western blots with anti-GLUT4 antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Translocation Assay, Western Blot, Glucose Assay, Membrane, Immunofluorescence, Staining

Journal: Ecotoxicology and environmental safety

Article Title: MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

doi: 10.1016/j.ecoenv.2021.112130

Figure Lengend Snippet: Fig. 6. Inhibition of miR-191 prevents arsenite-induced inhibition of the IRS1/AKT pathway and the translocation of GLUT4 in insulin-treated L-02 cells. L-02 cells were transfected with anti-miR-191 or anti-miR-NC (100 nM) for 24 h, then exposed to arsenite (0 or 4 μM) for 24 h, after which they were incubated with insulin (100 nM) for 30 min (A) The levels of miR-191 in L-02 cells were determined by qRT-PCR assays (mean ± SD, n = 3). (B) Glucose consumption and glycogen levels in L-02 cells were measured by glucose assay kits and glycogen assay kits, respectively (mean ± SD, n = 3). (C) Western blots of total protein were performed, and relative protein levels of p-IRS1, IRS1, and p-AKT in L-02 cells were measured (means ± SD, n = 3). (D) Western blots of membrane and cytoplasm proteins were performed, and relative protein levels of GLUT4 in L-02 cells were measured (mean ± SD, n = 3). (E) Immunofluorescence staining of GLUT4 and WGA in L-02 cells for the indicated groups were performed. Blue represents nuclear DNA staining by DAPI; green represents WGA staining; red represents GLUT4 staining. Bar = 50 µm. # p < 0.05, different from L-02 cells in the absence of arsenite; *p < 0.05, different from arsenite-exposed L-02 cells in the absence of anti-miR-191. (For inter pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The levels of GLUT4 were assessed by Western blots with anti-GLUT4 antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Translocation Assay, Transfection, Incubation, Quantitative RT-PCR, Glucose Assay, Western Blot, Membrane, Immunofluorescence, Staining

Journal: Ecotoxicology and environmental safety

Article Title: MicroRNA-191 blocking the translocation of GLUT4 is involved in arsenite-induced hepatic insulin resistance through inhibiting the IRS1/AKT pathway.

doi: 10.1016/j.ecoenv.2021.112130

Figure Lengend Snippet: Fig. 7. miR-191 via IRS1 is involved in arsenite-induced inhibition of the IRS1/AKT pathway and the translocation of GLUT4 in insulin-treated L-02 cells. L-02 cells were co-transfected with anti-miR-191 or anti-miR-NC (100 nM) and IRS1 siRNA or siRNA NC (100 nM) for 24 h, then exposed to arsenite (0 or 4 μM) for 24 h, after which they were incubated with insulin (100 nM) for 30 min. (A) Glucose consumption and glycogen levels in L-02 cells were measured by glucose assay kits and glycogen assay kits, respectively (mean ± SD, n = 3). (B) Western blots of total protein were performed, and relative protein levels of p-IRS1, IRS1, and p- AKT in L-02 cells were measured (mean ± SD, n = 3). (C) Western blots of membrane and cytoplasm protein were performed, and relative protein levels of GLUT4 in L-02 cells were measured (mean ± SD, n = 3). (D) For the indicated groups, immunofluorescence staining of GLUT4 and WGA in L-02 cells were performed. Blue represents nuclear DNA staining by DAPI; green represents WGA staining; red represents GLUT4 staining. Bar = 50 µm. # p < 0.05, different from L-02 cells in the absence of arsenite; *p < 0.05, different from arsenite-exposed L-02 cells in the absence of anti-miR-191; ꝉ p < 0.05, different from arsenite-exposed and anti-miR- 191-transfected L-02 cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The levels of GLUT4 were assessed by Western blots with anti-GLUT4 antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Translocation Assay, Transfection, Incubation, Glucose Assay, Western Blot, Membrane, Immunofluorescence, Staining

Journal: Frontiers in Physiology

Article Title: Involvement of atypical protein kinase C in the regulation of cardiac glucose and long-chain fatty acid uptake

doi: 10.3389/fphys.2012.00361

Figure Lengend Snippet: Expression of GLUT4, CD36, and VAMP2 in cardiomyocytes from wild-type and muscle-specific PKC-λ-knockout mice, and the effects of insulin and oligomycin treatment on phosphorylation of Akt, AS160, and ACC. Wild-type (WT) and PKC-λ-knockout (KO) cardiomyocytes were used to determine GLUT4, CD36, VAMP2, and GAPDH (loading control) expression, and after incubation for 20 min in the absence (Ctrl) or presence of 100 nM insulin (Ins) or 1 μM oligomycin (Oli) for assessment of phospho-Akt (Ser473), phospho-ACC (Ser79), and phospho-AS160 by Western blotting. In case of VAMP2, a sample from homogenized rat brain of identical protein content was used as a positive control on the expression of this protein. A representative Western blot is presented out of five experiments with different cardiomyocyte preparations.

Article Snippet: Antibodies against CD36 and GLUT4 were from Chemicon International Inc. (Temecula, USA).

Techniques: Expressing, Knock-Out, Incubation, Western Blot, Positive Control

Journal: Frontiers in Physiology

Article Title: Involvement of atypical protein kinase C in the regulation of cardiac glucose and long-chain fatty acid uptake

doi: 10.3389/fphys.2012.00361

Figure Lengend Snippet: Schematic presentation of the putative permissive role of atypical protein kinase C (isoforms ζ/λ) in GLUT4 and CD36 translocation in the heart. Upon binding to its receptor at the cell surface, insulin sequentially activates insulin-receptor substrate 2 (IRS2) and PI3K, resulting in the production of PIP 3 . Beyond PI3K, the insulin signal is split into two parallel branches. PIP 3 binds to both Akt/PKB and PKC-ζ/λ. In case of Akt/PKB this results in the activation and subsequent phosphorylation of AS160. In case of PKC-ζ/λ this does not result in further activation because the basally high levels of phosphatidic acid (PA) in the heart impose PKC-ζ/λ to be already bound to endosomal membranes in the absence of insulin treatment. Hence, PKC-ζ/λ maintains the endosomal membranes in a pre-active state, waiting for the insulin signal through activation of Akt/PKB and AS160 to complete the final stages in the budding of GLUT4- and CD36-containing vesicles for translocation to the sarcolemma. Upon contraction-induced activation of AMP-activated protein kinase (AMPK), a signaling cascade also involving ERK and PDK1, similarly will activate AS160 but not further activate PKC-ζ/λ. In agreement with the dependence of insulin-stimulated GLUT4- and CD36 translocation of PKC-ζ/λ, these processes will be subjected to inhibition by myristoylated PKC-ζ pseudosubstrate (ζ-PS). (Modified from Luiken et al., ).

Article Snippet: Antibodies against CD36 and GLUT4 were from Chemicon International Inc. (Temecula, USA).

Techniques: Translocation Assay, Binding Assay, Activation Assay, Inhibition, Modification

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Insulin stimulation fails to increase glucose uptake in human airway epithelia expressing F508del-CFTR. A: primary hBE cells were treated with bilateral insulin (black bars) for a total of 15 min in the presence of 2-deoxy-d-[3H]glucose to measure glucose uptake and retention within the cell layer. Cells were also treated with cytochalasin B (gray bars) to inhibit fusion of Glut4 storage vesicles (GSVs) to the plasma membrane. Unstimulated control cells are represented by white bars. NhBE cells responded to insulin by increasing their glucose uptake, whereas CFhBE cells did not respond to insulin. Cytochalasin B (CytoB) treatment prevented the full effect of insulin stimulation in NhBE cells but had no effect in CFhBE cells. B: comparably treated NuLi-1 and CuFi-5 cells had insulin stimulation responses similar to those seen in NhBE and CFhBE, respectively; n = 4 filters for each data point for each graph. C: temporal glucose uptake measurements indicated that CuFi-5 cells did not respond to treatment with bilateral insulin (■) and behaved similarly to unstimulated CuFi-5 cells (□) and unstimulated NuLi-1 cells (○) compared with stimulated NuLi-1 cells (●). D: NuLi-1 or CuFi-5 filters were pretreated with apical insulin solution for 15 min before measuring the uptake of 2-deoxy-d-[3H]glucose. The basolateral solution contained neither glucose nor insulin. Apical stimulation resulted in apical glucose uptake in NuLi-1 cells but not in CuFi-5 cells; n = 18 filters for each data bar. All data are shown as means ± SE where *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 by unprotected two-way ANOVA Fisher’s LSD test.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Expressing, Clinical Proteomics, Membrane, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Glut4 protein is detectable in human tracheal and bronchial epithelial cells by immunohistochemistry. Glut4 appears to be expressed by ciliated cells (black arrows) and by nonciliated cells (white arrows) in the human trachea (A) and bronchioles (C). Blood vessels (arrowheads) are located within 50–100 μm from the tracheal and bronchial epithelial cells. B, D, and F: control sections processed with nonspecific isotype control antibody. E: adipose tissue was used as a positive control tissue where Glut4 is known to be expressed.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Immunohistochemistry, Control, Positive Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Human airway epithelial cells express proteins necessary for insulin-stimulated glucose transport. A: quantitative RT (qRT)-PCR analysis of a subset of glucose transporter and related gene transcripts in freshly obtained human nasal epithelial (hNE) cells and air-liquid interface-cultured primary human bronchial epithelial (hBE) cells. White, freshly isolated hNE; gray, NhBE; hatched gray, cultured NuLi-1 cells; black, CFhBE; hatched black, cultured CuFi-5 cells. HKG, average of three housekeeping genes (ACTB/GAPDH/HPRT1). The official gene symbols and common names of the full qRT-PCR screen are given in Table 1. A cycle threshold (Ct) result of 35 is considered not expressed (broken line). Data are shown as means ± SE; n = 4 freshly isolated nasal epithelial scrapes and n = 3 cultured filters from 3 different donors. B: gel electrophoresis results demonstrate a primer dimer in glucose transporter (Glut) 4 PrimePCR amplification products (left), which may explain the high Ct values observed; an additional band below the expected size was detected in reactions not containing template. The remaining genes detected produced single bands as PrimePCR amplified gene products in the freshly isolated nasal curretage samples (right). C and D: immunoblots demonstrating protein expression of the insulin receptor, β-subunit (IR-β), and insulin-stimulated Glut4 in primary hBE cells (C), T84 colon carcinoma cells, and HeLa cervical adenocarcinoma cells and NuLi-1 and CuFi-5 human airway cells (D). E: immunoblots demonstrating Glut1 expression in NuLi-1 and CuFi-5 human airway cells compared with HEK293 cell lysates containing overexpressed Glut1-FLAG protein. F: immunoblots demonstrating Glut10 expression in NuLi-1 and CuFi-5 human airway cells compared with HEK293 cell lysates containing overexpressed Glut10-FLAG protein.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Quantitative RT-PCR, Cell Culture, Isolation, Nucleic Acid Electrophoresis, Amplification, Produced, Western Blot, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Gene list and qRT-PCR results from freshly isolated nasal epithelia and cultured bronchial epithelia

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Isolation, Cell Culture

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Primary and immortalized human airway epithelia exhibit apical organization of Glut4 storage vesicles. A and B: primary human airway epithelial cells without insulin stimulation show Glut4 (green) lateral to the nuclei (blue) in primary NhBE cells but show only apical organization relative to the nuclei in primary CFhBE cells. Apical β-catenin is pseudocolored in red to indicate cell borders. C and D: NuLi-1 and CuFi-5 cells showed prominent apical localization of Glut4 (green) relative to the nuclei (blue). Images were collected at ×60 magnification and generated with National Institutes of Health ImageJ software.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Generated, Software

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

doi: 10.1152/ajplung.00364.2016

Figure Lengend Snippet: Model for CFTR-mediated regulation of the airway glucose barrier. A: in normal airway epithelia without insulin stimulation, nutrients like glucose (red) slowly traverse the tight junction-regulated paracellular pathway (purple) while constitutive glucose transporters (blue) mediate baseline glucose metabolism and clearance from the paracellular space. Activity of CFTR (green) is normal, whereas Glut4 transporter (yellow) is stored in GSVs near the apical plasma membrane. B: airway epithelia stimulated with insulin decrease the rate of paracellular flux while simultaneously reducing the amount of glucose in the apical space through the activity of translocated Glut4 in the apical plasma membrane. C: in cystic fibrosis airway epithelia, the paracellular route is less stringent (dashed purple box) compared with normal epithelia. This results in more glucose being transported through the tight junction-mediated barrier, where the constitutive glucose transporters act to reduce the amount of glucose in the apical space. CFTR activity is reduced, effectively changing the fluid and ion balance of CF airway epithelia. D: in CF epithelia, insulin fails to activate GSV translocation, effectively reducing insulin-stimulated Glut4 activity while simultaneously allowing small molecules <10 kDa through the paracellular barrier, effectively allowing nutrient accumulation in the apical space.

Article Snippet: Antibodies used for immunoblotting include the following incubated overnight at room temperature, unless otherwise noted: rabbit monoclonal antibody (mAb) anti-human insulin receptor-β at 1:2,500 (no. 3025, 95 kDa; CST); mouse anti-actin at 1:20,000 (no. A5441, 47 kDa; Sigma) for 1 hour at room temperature (RT); rabbit anti-FLAG at 1:2,000 (no. F7425; Sigma); rabbit anti-human Glut4 at 1:2,500 (no. NBP1–49533, 54 kDa; Novus); rabbit anti-human SGLT1 at 1:1,000 (no. 07–1417, 72 kDa; Millipore); rabbit anti-human Glut1 at 1:1,000 (no. Ab15309, 54–60 kDa; Abcam); rabbit anti-human Glut10 at 1:1,000 (no. Ab33245, 52–60 kDa; Abcam); mouse anti-human panAKT at 1:1,000 (no. 2920, 60 kDa; CST); rabbit mAb anti-human Akt1 at 1:1,000 (no. 2938, 60 kDa; CST); rabbit mAb anti-human phospho-Akt1-S473 at 1:1,000 (no. 9018; CST); rabbit mAb anti-human Akt2 at 1:1,000 (no. 3063, 60 kDa; CST); rabbit mAb anti-human phospho-Akt2-S474 at 1:1,000 (no. 5899; CST); and mouse anti-human Akt3 at 1:1,000 (no. 8018, 60 kDa; CST).

Techniques: Activity Assay, Clinical Proteomics, Membrane, Translocation Assay