Immunohistochemical and in situ hybridization study of an insulin-like substance in fetal neuron cell cultures.
We studied the ability of fetal neuron cell cultures from different rabbit fetal brain gestational ages to produce and secrete an insulin-like substance (ILS). Neurons from 22-day gestation were incubated in serum-containing medium or insulin-free/serum-free medium, and 18-day gestation fetal rabbit neurons were also incubated in serum-free/insulin containing medium and serum-containing medium. The 22-day cultures survived in the serum-containing medium and the insulin-free/serum-free medium. The 18-day cultures died when incubated in the insulin-free/serum-free or serum-free/insulin-containing medium, but survived when incubated in serum-containing medium. Using immunohistochemical and in situ hybridization techniques, ILS and insulin-like mRNA were demonstrated within the 22-day cultures incubated in all media compositions, but not within the 18-day cultures incubated in the serum-containing medium. Ultrastructural studies of the 22-day cultures demonstrated an ILS in the endoplasmic reticulum, Golgi and cytoplasm. Northern blots showed the presence of an insulin-like mRNA within the 22-day gestation neuron cell cultures. Insulin receptor was present in the 22-day cultures, but was absent in the 18-day cultures. In addition, we characterized the ILS from the 22-day cultures incubated in the insulin-free/serum-free medium employing high-performance liquid chromatography (HPLC), radioimmunoassay and Western blots. Analysis by HPLC and Western blots demonstrated the presence of an ILS in the extract. We have shown that while 22-day fetal neuron cultures produce and secrete an insulin-like substance indistinguishable from authentic insulin, neuron cell cultures from early brain development do not express this capability.
Schechter
R
R
William K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, Tulsa 74136-7862.
Whitmire
J
J
Wheet
G S
GS
Beju
D
D
Jackson
K W
KW
Harlow
R
R
Gavin
J R
JR
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000196:embryology; D001923:Brain Chemistry / Q000502:physiology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007328:Insulin / Q000276:immunology / Q000378:metabolism*; D008854:Microscopy, Electron; D009474:Neurons / Q000378:metabolism*; D009841:Oligonucleotides / Q000138:chemical synthesis; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D011863:Radioimmunoassay; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism
D007328:Insulin; D009841:Oligonucleotides; D012333:RNA, Messenger; D011972:Receptor, Insulin