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