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First published online 14 November 2002
doi: 10.1242/jcs.00178

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An insulin-related peptide expressed in 3T3L1 adipocytes is localized in GLUT4 vesicles and secreted in response to exogenous insulin, which augments the insulin-stimulated glucose uptake

¹ Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
² Department of Internal Medicine (III), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan

View larger version (28K): [in a new window]   Fig. 1. Scheme illustrating the hypothesis of insulin-induced insulin release system in adipocytes. If (pro)insulin produced in adipocytes is to be included in GLUT4 vesicles, then (pro)insulin would need to be released in parallel with exocytosis of the GLUT4 vesicle in response to exogenous insulin stimulation. This would require the mechanism by which insulin receptor signaling causes movement of the GLUT4 vesicle from the intracellular pool to the plasma membrane and subsequently amplifies the insulin action through its own receptor via the autocrine mechanism.

View larger version (99K): [in a new window]   Fig. 2. Cellular localization of (pro)insulin expressed in 3T3L1 adipocytes. (A) Colocalization of (pro)insulin and GLUT4 in the same vesicle. 3T3L1 adipocytes were infected with Adex1CA pchi at an MOI of 20 pfu/cell, then they were fixed with 2% PFA and immunostained with both rabbit polyclonal anti-GLUT4 and mouse monoclonal anti-insulin antibodies followed by respective secondary antibody treatment (GLUT4: FITC, insulin: rhodamine). Immunofluorescent staining was observed by confocal laser microscopy with band-pass filter. Bar, 5 µm. (B) (Pro)insulin and GLUT1 are differently distributed in 3T3L1 adipocytes. After cells were infected and fixed as in A, they were immunostained with both rabbit polyclonal anti-GLUT1 and monoclonal insulin antibodies as described in A. Antibody complexes were visualized with appropriate secondary antibodies coupled to rhodamine (GLUT1) and FITC (insulin). Note the different immunofluorescent staining pattern between the peripheral staining of GLUT1 (rhodamine) and vesicular distribution of (pro)insulin (FITC) in the same cell with confocal laser-microscopy.

View larger version (13K): [in a new window]   Fig. 3. Proinsulin secretion from 3T3L1 adipocytes harboring the preproinsulin gene. Two days after 3T3L1 adipocytes were infected with Adex1CA pchi, cells were preincubated for 1 hour under KRBG buffer without insulin, and they were challenged by 10-9-10-7 M bovine insulin for 1 hour. At the end of incubation, medium was collected and proinsulin was assayed by human proinsulin ELISA.

View larger version (18K): [in a new window]   Fig. 4. Processing of proinsulin in 3T3L1 adipocytes. (A) Proinsulin and insulin separated by a peptide column. 3T3L1 adipocytes expressing the human insulin gene were labeled with 35S-Met/Cys for 1 hour, then chased for 1 hour. Cells were disrupted, and cell lysates were immunoprecipitated with an anti-insulin antibody. The immunoprecipitant was separated using a superdex peptide column, and radioactivity in each fraction was counted. (B) Reverse phase HPLC analysis of authentic recombinant human insulin and bovine insulin. The authentic insulin standards (10 µg in 100 µl 3 M acetic acid) were applied to the HPLC system with an acetonitrile gradient and monitored by 210 nm. The elution times for human and bovine insulin are 21 and 19 minutes, respectively. (C) Mature insulin in 3T3L1 adipocytes cells detected by HPLC. 3T3L1 adipocytes expressing the human insulin gene were disrupted by sonication, then cell lysates were analyzed by HPLC. Each fraction from the HPLC was neutralized and assayed using a human insulin ELISA kit.

View larger version (33K): [in a new window]   Fig. 5. Time-lapse confocal imaging of vesicles containing GFP-insulin in 3T3L1 adipocytes. 3T3L1 adipocytes were infected with Adex1CA pchi-GFP, 2 days later, time-lapse confocal imaging was performed. (A) Sequential images of 3T3L1 adipocytes expressing insulin-GFP. 3T3L1 adipocytes were stimulated by 10-7 M insulin, and each image was acquired every minute by setting the confocality to 5 µm. Typical confocal images were represented. (B) Change in the total number of GFP-labeled vesicles during stimulation was plotted against time (n=5 cells). The bars were expressed as a percentage of initial count of vesicle number prior to insulin stimulation.

View larger version (21K): [in a new window]   Fig. 6. Glucose uptake by 3T3L1 adipocytes. 3T3L1 adipocytes were infected with either Adex1CA pchi or Adex1w (for control), 2 days later, cells were preincubated for 1 hour under Hanks' solution without insulin and treated with different concentrations of insulin for 30 minutes at 37°C. Then, glucose uptake assay was performed over 20 minutes at room temperature per 1x106 cells under 10-9 M10-7 M insulin using 14C-2deoxy-D-glucose as a tracer (n=4). Data are means±s.e.m.