Journal: BMJ Open Diabetes Research & Care

Article Title: Huntingtin-associated protein 1 plays an essential role in the pathogenesis of type 2 diabetes by regulating the translocation of GLUT4 in mouse adipocytes

doi: 10.1136/bmjdrc-2020-001199

Figure Lengend Snippet: Regulation of glucose transporter isoform 4 (GLUT4) translocation in adipose tissue by huntingtin-associatedprotein 1 (HAP1). (A) Immunofluorescence staining of GLUT4 (left) with cultured primary adipocytes from wild-type (WT) and Hap1 -/- mice after 30 min insulin stimulation following 2-hour starvation and line-scan quantifications for the indicated lines drawn across the cells (right). The GLUT4 signal on the cell membrane is indicated by the black arrow. Scale bar, 20 µm. (B) The proportion of primary adipocytes with different GLUT4 immunofluorescence distribution at basal state and after 30 min insulin (INS) stimulation (n=24 for Hap1 -/- cells and n=56 for WT cells) (χ 2 test p<0.0001). (C) Detection of membrane-bound GLUT4 in primary Hap1 -/- and WT adipocytes after 30 min insulin stimulation following 2-hour starvation by western blotting. Na/K-ATPase was used as a membrane loading control. Data are presented as mean±SEM from three independent experiments. **p<0.01 (unpaired two-tailed t tests). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The pre-cleared lysates (500 μg) were incubated with 1 μg of goat anti-HAP1 antibody (sc-12556; Santa Cruz, Dallas, Texas, USA) or goat anti-GLUT4 antibody (sc-1608, Santa Cruz) or rabbit anti-GLUT4 antibody (ab654; Abcam, Cambridge, UK) or rabbit anti-sortilin antibody (ab16640, Abcam) at 4°C for 12 hours, and then incubated with protein A/G beads for 1 hour.

Techniques: Translocation Assay, Immunofluorescence, Staining, Cell Culture, Membrane, Western Blot, Control, Two Tailed Test

Journal: BMJ Open Diabetes Research & Care

Article Title: Huntingtin-associated protein 1 plays an essential role in the pathogenesis of type 2 diabetes by regulating the translocation of GLUT4 in mouse adipocytes

doi: 10.1136/bmjdrc-2020-001199

Figure Lengend Snippet: Huntingtin-associatedprotein 1 (HAP1) interacted with glucose transporterisoform 4 (GLUT4) in vivo and in vitro. (A) HAP1 and GLUT4 were colocalized in an intracellular location. Top row: HAP1-CFP and GLUT4-mCherry cotransfected HEK293 cells. Scale bar, 10 µm. Middle row: Double staining of mouse adipose tissue with HAP1 (green) and GLUT4 (red). Scale bar, 100 µm. Bottom row: Double staining of wild-type (WT) primary adipocytes with HAP1 (green) and GLUT4 (red). Scale bar, 20 µm. The nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). (B) Co-immunoprecipitation (Co-IP) assay was applied for identification of the interaction between HAP1 and GLUT4. Detection of HAP1 and GLUT4 in HEK293 cells cotransfected with HAP1 and GLUT4 plasmids (left) and mouse adipose tissue (right) by western blotting. Lanes 1 and 4 for the positive control of total protein lysate, lanes 2 and 5 for the IgG as negative control, and lanes 3 and 6 for the Co-IP sample. (C) Co-IP was applied for the identification of endogenous HAP1 and GLUT4 expression of HEK293 cells. HEK293 cells were with or without HAP1 or GLUT4 plasmid, and the other protein was detected by western blotting. Lane 1 for the positive control of HEK293 cells cotransfected with HAP1 and GLUT4 plasmids’ total protein lysate, lane 2 for total protein lysate of sample for Co-IP, lane 3 for the IgG as negative control and lane 4 for the Co-IP sample. IB, immunoblotting; IP, immunoprecipitation; WAT, white adipose tissue.

Article Snippet: The pre-cleared lysates (500 μg) were incubated with 1 μg of goat anti-HAP1 antibody (sc-12556; Santa Cruz, Dallas, Texas, USA) or goat anti-GLUT4 antibody (sc-1608, Santa Cruz) or rabbit anti-GLUT4 antibody (ab654; Abcam, Cambridge, UK) or rabbit anti-sortilin antibody (ab16640, Abcam) at 4°C for 12 hours, and then incubated with protein A/G beads for 1 hour.

Techniques: In Vivo, In Vitro, Double Staining, Staining, Co-Immunoprecipitation Assay, Western Blot, Positive Control, Negative Control, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: BMJ Open Diabetes Research & Care

Article Title: Huntingtin-associated protein 1 plays an essential role in the pathogenesis of type 2 diabetes by regulating the translocation of GLUT4 in mouse adipocytes

doi: 10.1136/bmjdrc-2020-001199

Figure Lengend Snippet: Huntingtin-associatedprotein 1 (HAP1) formed a protein complex with glucose transporterisoform 4 (GLUT4) via sortilin. (A) Co-immunoprecipitation (Co-IP) was applied for identification of the interaction among HAP1, GLUT4 and sortilin. Detection in HEK293 cells cotransfected with HAP1 and GLUT4 plasmids (left) and mouse adipose tissue (right) by western blotting. (B) Sortilin protein expression was detected after 48-hour interference. (C) HEK293 cells were transfected with HAP1 plasmids and interfered with or without si-Sortilin, and proteins were collected for Co-IP. (D) HEK293 cells were transfected with GLUT4 plasmids and interfered with or without si-Sortilin, and proteins were collected for Co-IP. (E) HEK293 cells were cotransfected with HAP1 and GLUT4 plasmids; meanwhile, sortilin was reduced by small interfering RNA (siRNA). Interaction between HAP1 and GLUT4 was detected in sortilin-siRNA or sortilin-scramble-treated HEK293 cells. Lanes 1 and 4 for the positive control of total protein lysate, lanes 2 and 5 for the IgG as negative control, and lanes 3 and 6 for the Co-IP sample. (F) The schematic model and proposed mechanism of HAP1, GLUT4 and sortilin–protein complex involved in the insulin-stimulated GLUT4 translocation. In mouse adipocytes, HAP1 forms a complex with GLUT4 via sortilin and interacts with motor proteins, promoting the GLUT4 transport along cytoskeleton. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IB, immunoblotting; IP, immunoprecipitation; KIF5B, kinesin family motorprotein 5; MYO5, myosin 5; NC, negative control; WAT, white adipose tissue.

Article Snippet: The pre-cleared lysates (500 μg) were incubated with 1 μg of goat anti-HAP1 antibody (sc-12556; Santa Cruz, Dallas, Texas, USA) or goat anti-GLUT4 antibody (sc-1608, Santa Cruz) or rabbit anti-GLUT4 antibody (ab654; Abcam, Cambridge, UK) or rabbit anti-sortilin antibody (ab16640, Abcam) at 4°C for 12 hours, and then incubated with protein A/G beads for 1 hour.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Expressing, Transfection, Small Interfering RNA, Positive Control, Negative Control, Translocation Assay

Journal: Pharmaceuticals

Article Title: Andrographolide Promotes Uptake of Glucose and GLUT4 Transport through the PKC Pathway in L6 Cells

doi: 10.3390/ph15111346

Figure Lengend Snippet: AP-EtOAc promoted the uptake of glucose and enhanced GLUT4 transport in L6 cells. ( A ) Values on the Y-axis represent the fold relationship of glucose uptake by L6 cells compared with that in normal conditions. Uptake of glucose detected using a Glucose oxidase kit in L6 cells, the data represent the mean ± s.e.m. of values from three separate experiments. The control group was considered as 1 for data analysis. ( B ) Values on the Y-axis represent the fold relationship of glucose uptake by L6 cells compared with that in normal conditions. Uptake of glucose in L6 cells induced by AP-EtOAc in insulin resistant L6 cells, the data represent the mean ± s.e.m. of values from three separate experiments. ( C ) Images of 60 μg/mL AP-EtOAc stimulating GLUT4 transport in L6 cells. Scale bar = 50 μm. ( D ) Calculation of fluorescence intensity in myc-GLUT4-mOrange-L6 cells using Zen 2010 software, n = 30 cells. *: p < 0.05; **: p < 0.01, ***: p < 0.001.

Article Snippet: GLUT4 (1F8) Mouse mAb (anti-GLUT4, Cat# 2213S), β-Actin (8H10D10) Mouse mAb (anti-β-actin, Cat# 3700S), Phospho-Akt (Ser473) (193H12) Rabbit mAb (anti-p-Akt, Cat# 4058S), Akt Rabbit Ab (Anti-Akt, Cat# 9272S), Phospho-AMPKα (Thr172) (D79.5E) Rabbit mAb (anti-p-AMPK, Cat# 4188S), AMPKα Rabbit Ab (anti-AMPK, Cat# 2532S), and Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit mAb (anti-p-PKC, Cat# 2060S) were the products of Cell Signaling Technology (Boston, MA, USA).

Techniques: Control, Fluorescence, Software

Journal: Pharmaceuticals

Article Title: Andrographolide Promotes Uptake of Glucose and GLUT4 Transport through the PKC Pathway in L6 Cells

doi: 10.3390/ph15111346

Figure Lengend Snippet: AP-EtOAc enhanced GLUT4 protein expression and GLUT4 fusion with the plasma membrane in L6 cells. ( A ) FITC fluorescence assay in myc-GLUT4-mOrange cells treated with 60μg/mL AP-EtOAc or 100 nM insulin. Scale bar: 5 μm in single cell image, 50 μm in multi-cell image. ( B ) Number of GLUT4-mOrange-positive cells, the data represent the mean ± s.e.m. of values from three separate experiments with between 200 and 300 cells being examined in each experiment. ( C ) The expression level of GLUT4 after treated with 100 nM insulin or different concentrations of AP-EtOAc for 30 min in L6 cells. The control group was considered as 1 for data analysis. The data represent the mean ± s.e.m. of values from three separate experiment. *: p < 0.05; **: p < 0.01, ***: p < 0.001.

Article Snippet: GLUT4 (1F8) Mouse mAb (anti-GLUT4, Cat# 2213S), β-Actin (8H10D10) Mouse mAb (anti-β-actin, Cat# 3700S), Phospho-Akt (Ser473) (193H12) Rabbit mAb (anti-p-Akt, Cat# 4058S), Akt Rabbit Ab (Anti-Akt, Cat# 9272S), Phospho-AMPKα (Thr172) (D79.5E) Rabbit mAb (anti-p-AMPK, Cat# 4188S), AMPKα Rabbit Ab (anti-AMPK, Cat# 2532S), and Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit mAb (anti-p-PKC, Cat# 2060S) were the products of Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Clinical Proteomics, Membrane, Fluorescence, Control

Journal: Pharmaceuticals

Article Title: Andrographolide Promotes Uptake of Glucose and GLUT4 Transport through the PKC Pathway in L6 Cells

doi: 10.3390/ph15111346

Figure Lengend Snippet: AND promoted GLUT4 expression and fusion into plasma membrane. ( A ) FITC fluorescence analysis of L6 cells treated with AND or Insulin. Scale bar: 10 μm for single-cell images and 50 μm for multi-cell images. ( B ) The number of GLUT4-mOrange-positive cells, the data represent the mean ± s.e.m. of values from three separate experiments with between 200 and 300 cells examined in each experiment. ( C ) The expression level of GLUT4 after treatment with 100 nM insulin or different concentrations of AND for 30 min in L6 cells. The control group was considered as 1 for data analysis. The data represent the mean ± s.e.m. of values from three separate experiments. *: p < 0.05; **: p < 0.01; ****: p < 0.0001.

Article Snippet: GLUT4 (1F8) Mouse mAb (anti-GLUT4, Cat# 2213S), β-Actin (8H10D10) Mouse mAb (anti-β-actin, Cat# 3700S), Phospho-Akt (Ser473) (193H12) Rabbit mAb (anti-p-Akt, Cat# 4058S), Akt Rabbit Ab (Anti-Akt, Cat# 9272S), Phospho-AMPKα (Thr172) (D79.5E) Rabbit mAb (anti-p-AMPK, Cat# 4188S), AMPKα Rabbit Ab (anti-AMPK, Cat# 2532S), and Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit mAb (anti-p-PKC, Cat# 2060S) were the products of Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Clinical Proteomics, Membrane, Fluorescence, Control

Journal: Pharmaceuticals

Article Title: Andrographolide Promotes Uptake of Glucose and GLUT4 Transport through the PKC Pathway in L6 Cells

doi: 10.3390/ph15111346

Figure Lengend Snippet: AND induced GLUT4 expression through the PKC signaling pathway. The control group was considered as 1 for data analysis. The expression of GLUT4 induced by AND under the action of three inhibitors. ns: p >0.05; *: p < 0.05; **: p < 0.01.

Article Snippet: GLUT4 (1F8) Mouse mAb (anti-GLUT4, Cat# 2213S), β-Actin (8H10D10) Mouse mAb (anti-β-actin, Cat# 3700S), Phospho-Akt (Ser473) (193H12) Rabbit mAb (anti-p-Akt, Cat# 4058S), Akt Rabbit Ab (Anti-Akt, Cat# 9272S), Phospho-AMPKα (Thr172) (D79.5E) Rabbit mAb (anti-p-AMPK, Cat# 4188S), AMPKα Rabbit Ab (anti-AMPK, Cat# 2532S), and Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit mAb (anti-p-PKC, Cat# 2060S) were the products of Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Control

Journal: Pharmaceuticals

Article Title: Andrographolide Promotes Uptake of Glucose and GLUT4 Transport through the PKC Pathway in L6 Cells

doi: 10.3390/ph15111346

Figure Lengend Snippet: Translocation of GLUT4 and uptake of glucose induced by AND in L6 cells blocked by 0 mM extracellular Ca 2+ and 0 mM extracellular Ca 2+ + 10 μM BAPTA-AM. ( A ) L6 cells were stimulated by AND under the conditions of 2 mM extracellular Ca 2+ , 0 mM extracellular Ca 2+ , and 0 mM extracellular Ca 2+ + 10 μM BAPTA-AM. The distribution of red and green fluorescence in L6 cells was detected by confocal laser scanning microscopy. Scale bar: 10 μm for single-cell images and 50 μm for multi-cell images. ( B ) The number of GLUT4-mOrange-positive cells data represent the mean ± s.e.m. of values from three separate experiments, with between 200 and 300 cells examined in each experiment. ( C ) Uptake of glucose induced by insulin or AND in the three Ca 2+ buffering systems. The three groups on the left are the control group, and the three groups on the right represent glucose uptake after adding insulin and AND. The data represent the mean ± s.e.m. of values from three separate experiments. ns: p > 0.05; *: p < 0.05; **: p < 0.01; ****: p < 0.0001.

Article Snippet: GLUT4 (1F8) Mouse mAb (anti-GLUT4, Cat# 2213S), β-Actin (8H10D10) Mouse mAb (anti-β-actin, Cat# 3700S), Phospho-Akt (Ser473) (193H12) Rabbit mAb (anti-p-Akt, Cat# 4058S), Akt Rabbit Ab (Anti-Akt, Cat# 9272S), Phospho-AMPKα (Thr172) (D79.5E) Rabbit mAb (anti-p-AMPK, Cat# 4188S), AMPKα Rabbit Ab (anti-AMPK, Cat# 2532S), and Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit mAb (anti-p-PKC, Cat# 2060S) were the products of Cell Signaling Technology (Boston, MA, USA).

Techniques: Translocation Assay, Fluorescence, Confocal Laser Scanning Microscopy, Control

Journal: Metabolites

Article Title: Altered Pulmonary Glucose Transport Is Restored by Metformin Treatment in an Obese Type 2 Diabetic Animal Model

doi: 10.3390/metabo15110717

Figure Lengend Snippet: Type 2 diabetes (T2Dx) induced decreased pulmonary protein expression of the class I glucose transporter (GLUT), which was partially rescued by in vivo metformin (Met) treatment. Total protein expression of ( A ) GLUT1, ( B ) GLUT2, and ( C ) GLUT4 in the whole lung of control (con), untreated T2Dx, and metformin-treated T2Dx mice. Top panels: representative Western blot from total lysate of the whole lung; loading control: beta actin. Bottom panels: Mean ± SE of total GLUT protein content (values normalized to β-actin and its respective controls, n = 4–6/group). * p < 0.05 vs. control. Methods: Western blotting.

Article Snippet: Primary antibodies used included: polyclonal rabbit anti-mouse GLUT1 (1:500, Abcam #ab652), GLUT2 (1:500, Santa Cruz #sc-7582), GLUT3 (1:500, Abcam #ab54460), GLUT4 (1:750, AbD Serotec #4670-1704), GLUT8 (1:500, Bioss #bs4241R), GLUT10 (1:750, Thermo Fisher #PA1-46137), and GLUT12 (1:500, Abcam #ab75441). β-actin was detected using a mouse monoclonal antibody (Santa Cruz #sc-47778).

Techniques: Expressing, In Vivo, Control, Western Blot

Journal: Metabolites

Article Title: Altered Pulmonary Glucose Transport Is Restored by Metformin Treatment in an Obese Type 2 Diabetic Animal Model

doi: 10.3390/metabo15110717

Figure Lengend Snippet: Type 2 diabetes (T2Dx) decreased pulmonary cell surface glucose transporter (GLUT) protein expression, which was rescued by in vivo metformin (Met) treatment. Cell-surface protein expression of ( A ) GLUT4 and ( B ) GLUT8 in whole lung of control (Con), untreated T2Dx, and metformin-treated T2Dx animals. Top panels: representative Western blot. Bottom panels: Mean ± SE of cell surface GLUT protein content (values normalized to respective controls, n = 4/group). L: Labeled (cell surface fraction), UL: unlabeled (intracellular fraction). * p < 0.05 vs. control, via one-way ANOVA. Methods: biotinylated photolabeling assay.

Article Snippet: Primary antibodies used included: polyclonal rabbit anti-mouse GLUT1 (1:500, Abcam #ab652), GLUT2 (1:500, Santa Cruz #sc-7582), GLUT3 (1:500, Abcam #ab54460), GLUT4 (1:750, AbD Serotec #4670-1704), GLUT8 (1:500, Bioss #bs4241R), GLUT10 (1:750, Thermo Fisher #PA1-46137), and GLUT12 (1:500, Abcam #ab75441). β-actin was detected using a mouse monoclonal antibody (Santa Cruz #sc-47778).

Techniques: Expressing, In Vivo, Control, Western Blot, Labeling