Ethidium Bromide-induced Inhibition of Mitochondrial Gene Transcription Suppresses Glucose-stimulated Insulin Release in the Mouse Pancreatic β-Cell Line βHC9*

Recently, a mitochondrial mutation was found to be associated with maternally 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., Horai, 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 have investigated the involvement of mitochondrial function in insulin secretion. Culture of the pancreatic β-cell line, βHC9, with low dose ethidium bromide (EB) (0.4 μg/ml) for 2–6 days resulted in a substantial decrease in the transcription level of mitochondrial DNA (to 10–20% of the control cells) without changing its copy number, whereas the transcription of nuclear genes was grossly unaffected. Electron microscopic analysis revealed that treatment by EB caused morphological changes only in mitochondria and not in other organelles such as nuclei, endoplasmic reticula, Golgi bodies, or secretory granules. When the cells were treated with EB for 6 days, glucose (20 mm) could no longer stimulate insulin secretion, while glibenclamide (1 μm) still did. When EB was removed after 3- or 6-day treatment, mitochondrial gene transcription recovered within 2 days, and the profiles of insulin secretion returned to normal within 7 days. Studies with fura-2 indicated that in EB-treated cells, glucose (20 mm) failed to increase intracellular Ca2+, while the effect of glibenclamide (1 μm) was maintained. Our system provides a unique way to investigate the relationship between mitochondrial function and insulin secretion.

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