Immunoreactive insulin from mouse brain cells in culture and whole rat brain

Abstract

Foetal mouse brain cells were cultured as described previously [Sotelo, Gibbs, Gajdusek, Toh & Wurth (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 653-657] without added insulin and without foetal calf serum after 12 days in culture. Examination by phase-contrast microscopy showed that these modifications did not appear to affect growth and development of the cells adversely. Silver impregnation of the cultures and indirect immunofluorescence following reaction with tetanus toxin showed that a high proportion of the cells resembled neurones. Analysis of concentrated culture medium by radioimmunoassay and high-pressure liquid chromatography (h.p.l.c.) revealed that the cells produced two main forms of immunoreactive insulin which differed from authentic pancreatic insulin in retention time. Immunoreactive somatostatin was also produced in culture and this was resolved into at least three forms by h.p.l.c. Immunoreactive insulin was also extracted from whole rat brain by using two published procedures. The method of Havrankova, Schmechel, Roth & Brownstein [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 5737-5741] consistently gave greater yields of insulin than did that of Eng & Yalow [Diabetes (1980) 29, 105-109] and the concentration was about three times that of plasma. The extracted insulin was further characterized by h.p.l.c. in each case and was found to behave like authentic pancreatic insulin. The production of insulin and somatostatin by foetal mouse brain cells in culture suggests that they may be a useful model system for studies of neuropeptide biosynthesis.