Ethidium bromide-induced inhibition of mitochondrial gene transcription suppresses glucose-stimulated insulin release in the mouse pancreatic beta-cell line betaHC9.
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 beta-cell line, betaHC9, with low dose ethidium bromide (EB) (0.4 microg/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 microM) 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 microM) was maintained. Our system provides a unique way to investigate the relationship between mitochondrial function and insulin secretion.
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
T
T
Hayakawa
M
M
Noda
K
K
Yasuda
H
H
Yorifuji
S
S
Taniguchi
I
I
Miwa
H
H
Sakura
Y
Y
Terauchi
J
J
Hayashi
G W
GW
Sharp
Y
Y
Kanazawa
Y
Y
Akanuma
Y
Y
Yazaki
T
T
Kadowaki
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000235:genetics*; D004996:Ethidium / Q000494:pharmacology*; D016257:Fura-2 / Q000378:metabolism; D005941:Glucokinase / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D005905:Glyburide / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects*; D051379:Mice; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000648:ultrastructure; D010766:Phosphorylation; D012333:RNA, Messenger / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects*
D004272:DNA, Mitochondrial; D007004:Hypoglycemic Agents; D007328:Insulin; D012333:RNA, Messenger; D006593:Hexokinase; D005941:Glucokinase; D004996:Ethidium; D005947:Glucose; D005905:Glyburide; D002118:Calcium; D016257:Fura-2