In pancreatic β-cells, mitochondria are of particular importance in the regulation of insulin secretion because they produce ATP as well as other coupling factors which link nutrient metabolism and insulin exocytosis [11
]. mtDNA-depleted β-cell lines show complete absence of nutrient-stimulated insulin secretion [21
]. Patients with mtDNA mutations develop diabetes, accounting for up to 1% of the total number of diabetic patients [22
]. Moreover, postmortem islets from type 2 diabetes patients display functional deterioration of mitochondria [23
]. Therefore, factors that disturb the mitochondrial function in pancreatic β-cells might affect metabolism-secretion coupling and diabetogenesis.
The present study showed that the effective anti-HBV agent clevudine has a negative effect on the copy number and transcription of mtDNA in insulin-releasing cells and hepatoma cells. The reduced expressions of mtDNA-encoded proteins lead to attenuation of mitochondrial function. In insulin-releasing cells, clevudine-induced mitochondrial dysfunction can elicit defective insulin secretion in response to substrates for mitochondrial metabolism. To our knowledge, this is the first demonstration that an antiviral agent can impair nutrient-stimulated insulin secretion as a result of mitochondrial dysfunction. Because of their high dependency on mitochondrial function in metabolism-secretion coupling, insulin-secreting cells provide a useful model to investigate the functional consequences of drug-induced mitochondrial toxicity.
NRTIs are widely used to treat various viral diseases such as acquired immunodeficiency syndrome (AIDS) and hepatitis B [24
]. However, in vitro
studies showed that NRTIs can alter mtDNA content by inhibiting DNA polymerase-γ [25
]. Moreover, myopathy accompanied by mtDNA depletion has been reported in NRTI-treated patients [4
]. Clevudine treatment has also been associated with the development of mitochondrial complications. In contrast to early studies [2
], depletion of mtDNA in skeletal muscle has been observed in patients treated with clevudine [7
]. Typical histological features of mitochondrial myopathy and abnormal mitochondrial morphology were displayed in tissues from patients with increased lactate dehydrogenase and lactate levels [8
]. Although the incidence of clevudine-induced myopathy was reported to be low (~5%) [9
], a substantial proportion (~14.5%) of clevudine-treated patients have been found to experience symptoms, signs, and laboratory abnormalities relevant to clevudine-induced myopathy [27
To directly confirm the effects of clevudine on mitochondrial function, we cultured cells with medium containing different concentrations of clevudine for 4 weeks. Clevudine markedly decreased the MTT signal without significant changes in cellular protein implying the diminished enzyme activities for reduction of MTT. Since MTT assay is not specific to evaluate mitochondrial function, measurement of oxygen consumption rate or citrate synthase activity could provide more concrete evidence to prove the mitochondrial defects. Consistent with mtDNA depletion, COX activity and cellular ATP content were reduced. Decreased mitochondrial fatty acid oxidation could induce triglyceride accumulation [19
]. To avoid lipotoxic effects of palmitate in insulin-secreting cells [28
], we loaded unsaturated fatty acid oleate for 24 hours, which elicited a marked increase of lipid accretion within clevudine-treated cells. The inhibitory effect of clevudine on insulin secretion was more sensitive than the effect on ATP level. We can speculate that the treatment of 100 μM clevudine elicited significant reduction of ATP/ADP ratio which is the main signal for closing ATP-sensitive K+
channel and insulin exocytosis.
We also observed some compensatory responses to reduced mtDNA copy number and its functional consequences. First, PGC-1α and its downstream transcriptional factors, NRF-1 and Tfam, were upregulated by clevudine. Second, nuclear DNA-encoded succinate dehydrogenase was also upregulated, which has already been observed in muscle of patients suffering from clevudine-induced myopathy [10
]. Third, lactate production was modestly increased in association with diminished ATP content. Pancreatic β-cells and clonal β-cell lines are known to have very low lactate dehydrogenase levels, which contribute to their dependency on mitochondrial function. The increase in lactate production observed in our study also demonstrates that clevudine imposes selective defects on mitochondria rather than overall cytotoxicity.
In our study, mtDNA copy number in clevudine (1 mM)-treated cells was decreased to half of that in control. It has been reported, however, that to evoke mitochondrial dysfunction mtDNA level should fall below 60% which was named as 'phenotypic threshold' [29
]. This can be explained by genetic and functional complementation at the levels of transcription, translation, enzyme activity and cell activity. Several investigators showed that NRTI such as zidovudine and stavudine can also induce mitochondrial dysfunction independent from lack of mtDNA [20
]. Thus, we cannot exclude the possibility that clevudine could be involved in multiple site of inhibition of mitochondrial function in addition to the effects of mtDNA depletion.
Niu et al. [31
] suggested that the intracellular level of the triphosphate form of clevudine in cells exposed to 1 μM extracellular clevudine approximates the plasma level in patients receiving a 30 mg dose. Our results indicated that impairments in mitochondrial function and insulin secretion are elicited only by high concentrations of clevudine (> 100 μM). This means that clevudine would minimally affect mitochondrial function within the therapeutic concentration range. It is noteworthy, however, that mutations or polymorphisms of DNA polymerase-γ were identified in NRTI-treated patients with mitochondrial complications [32
]. This suggests that genetic alterations in DNA polymerase-γ are not normally deleterious, but that certain conditions such as NRTI treatment may push mitochondrial activity below the clinical threshold, causing pathogenic dysfunction [33
]. Differences in genetic susceptibility to mitochondrial toxicity could be one explanation for why a limited proportion of patients receiving clevudine have complications including myopathy.
Clevudine-induced depletion of mtDNA is not restricted to insulin-secreting cells but is also observed in cultured hepatoma cells or muscle tissue from patients [7
]. Mitochondrial dysfunction in insulin target tissues such as liver and muscle could result in insulin resistance and diabetes [34
]. In addition to defects in insulin secretion, decreased sensitivity in insulin target cells can also participate in diabetogenesis in patients receiving clevudine who might have a high susceptibility to mitochondrial toxicity. Interestingly, several reports have shown that NRTI induces intramitochondrial pyrimidine deficiency which may aggravate mtDNA depletion and mitochondrial dysfunction [35
]. They also discovered that uridine supplementation attenuates steatohepatitis or mitochondrial myopathy induced by NRTI. Further studies concerning the effects of NRTIs on mitochondrial function in different cell types may help us understanding these intractable complications and develop novel antiviral agents.