ETHIDIUM BROMIDE-INDUCED INHIBITION OF MITOCHONDRIAL GENE-TRANSCRIPTION SUPPRESSES GLUCOSE-STIMULATED INSULIN RELEASE IN THE MOUSE PANCREATIC BETA-CELL LINE BETA-HC9

Recently, a mitochondrial mutation was found to be associated with mat ernally inherited diabetes mellitus (Kadowaki, T., Kadowaki, H., Mori, Y., Tobe, K., Sakuta, R., Suzuki, Y., Tanabe, Y, Sakura, H., Awata, T ., Goto, Y., Hayakawa, T., Matsuoka, K., Kawamori, R., Kamada, T., Hor ai, S., Nonaka, I., Hagura, R., Akanuma, Y., and Yazaki, Y. (1994) N. Engl. J. Med. 330, 962-968). In order to elucidate its etiology, we ha ve investigated the involvement of mitochondrial function in insulin s ecretion. Culture of the pancreatic beta-cell line, beta HC9, with low dose ethidium bromide (EB) (0.4 mu g/ml) for 2-6 days resulted in a s ubstantial decrease in the transcription level of mitochondrial DNA (t o 10-20% of the control cells) without changing its copy number, where as the transcription of nuclear genes was grossly unaffected. Electron microscopic analysis revealed that treatment by EB caused morphologic al changes only in mitochondria and not in other organelles such as nu clei, endoplasmic reticula, Golgi bodies, or secretory granules. When the cells were treated with EB for 6 days, glucose (20 mM) could no lo nger stimulate insulin secretion, while glibenclamide (1 mu M) Still d id. When EB was removed after 3- or B-day treatment, mitochondrial gen e transcription recovered within 2 days, and the profiles of insulin s ecretion returned to normal within 7 days. Studies with fura-2 indicat ed that in EB-treated cells, glucose (20 mM) failed to increase intrac ellular Ca2+ while the effect of glibenclamide (1 mu M) was maintained . Our system provides a unique way to investigate the relationship bet ween mitochondrial function and insulin secretion.