To determine whether mtDNA and mitochondrial respiratory function in pancreatic beta cells are necessary for the phenotypic expression of glucose-stimulated insulin secretion, we used a cultured mouse pancreatic beta cell line, MIN6, and two derivative lines, mtDNA knockout MIN6 ( 0 MIN6) and mtDNA repopulated cybrid MIN6. The MIN6 cells retain the property of glucose-stimulated insulin secretion, but their mtDNA knockout induced the loss of mitochondrial transcription, translation, and respiration activity, without inhibition of transcription of the insulin gene or loss of succinate dehydrogenase activity, indicating that the observed mitochondrial dysfunction in 0 MIN6 cells was not due to a cytotoxic side effect derived from the mtDNA knockout. Moreover, the mtDNA depletion also inhibited both the glucose-stimulated increase in the intracellular free Ca 2؉ content and the elevation of insulin secretion. The possibility of the involvement of nuclear genome-encoded factors in this process was excluded by the observation that the missing sensitivity to extracellular glucose stimulation in 0 MIN6 cells was restored reversibly by repopulation with foreign mtDNA and isolating cybrid MIN6 clones. Therefore, these findings provide unambiguous evidence for the involvement of the mitochondrial dysfunction induced by mtDNA impairment in developing pathogeneses of some forms of diabetes mellitus.

For isolation of mouse mtDNA-less ( 0 ) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting 0 mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the 0 cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells. With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice. Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (⌬mtDNA 5823 ) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.

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