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.
If an adipocyte is programmed to secrete insulin, then the insulin released may amplify the insulin action by an autocrine manner. To examine this hypothesis in vitro, we investigated the effects of expressing the preproinsulin gene in 3T3L1 adipocytes on (pro)insulin release and glucose uptake. The human preproinsulin gene was transferred into 3T3L1 adipocytes by infecting the cells with recombinant adenovirus Adex1CA human preproinsulin. Immunocytochemical studies showed that (pro)insulin is associated with vesicular structures that colocalize with GLUT4 vesicles but not with GLUT1 vesicles. We then examined insulin-induced proinsulin release from 3T3L1 adipocytes expressing the insulin gene. The exogenously administered insulin stimulated proinsulin release from these cells in a dose-dependent manner. HPLC determination revealed the existence of mature human insulin in these cells, which suggested the release of mature insulin into the medium. Further we monitored the (pro)insulin release from these cells with confocal laser microscopy using the expression of a fusion protein between insulin and green fluorescent protein (GFP). Time-lapse confocal laser-scanning microscopy revealed that the total number of vesicles containing insulin-GFP was decreased by the addition of 10(-7) M insulin within 1 minute. Finally, we examined the insulin-stimulated glucose uptake by these cells. The data showed that insulin-stimulated glucose uptake increased to about 150% of that of control cells in response to exogenously administered insulin, indicating that the insulin released augmented the insulin-stimulated glucose uptake in an autocrine manner. Thus, the data support our hypothesis, indicating that we could construct the insulin-regulated insulin release system in adipocytes by introducing the preproinsulin gene.
Nakamichi
Yoko
Y
Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Ohara-Imaizumi
Mica
M
Ishida
Hitoshi
H
Nagamatsu
Shinya
S
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D019898:Autocrine Communication / Q000235:genetics; D002451:Cell Compartmentation / Q000235:genetics; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology; D005456:Fluorescent Dyes; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008164:Luminescent Proteins; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism*; D011930:Reaction Time / Q000235:genetics; D011993:Recombinant Fusion Proteins; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*
D005456:Fluorescent Dyes; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; D049452:Green Fluorescent Proteins; D011384:Proinsulin; D005947:Glucose