Review for 10.1186/1471-230X-12-4 (2012)

Reviewer's report
Title:High-dose clevudine impairs mitochondrial function and glucose-stimulated
insulin secretion in INS-1E cells
Version: 1Date:29 August 2011
Reviewer: Bernard Fromenty
Reviewer's report:
The article submitted by Dr Jang and collaborators shows that high
concentrations of the anti-HBV nucleoside analogue clevudine induce
cytotoxicity, deplete mitochondrial DNA (mtDNA), reduce Cox-1 mRNA levels,
lower cellular ATP content and impair insulin release under high glucose
concentration, in the rat pancreatic beta cell line INS-1E. Moreover, high
concentrations of clevudine are able to decrease mitochondrial DNA (mtDNA)
and Cox-1 mRNA levels in the human hepatoma cell line HepG2. Although these
findings are interesting and novel, there are several important issues which need
to be addressed in order to strengthen the manuscript.
Major issues (major compulsory revisions):
1- Statistical analysis is not appropriate since the Student’s t test is used to
compare two groups of data. Because the authors are comparing several groups,
a one-way ANOVA must be used (Figure 1 and 2A). Moreover, when the effects
of clevudine and glucose levels are simultaneously studied, a two-way ANOVA
must be employed.
2- The data regarding the effect of 100 micromolar clevudine on PGC-1alpha,
Tfam and Nrf1 expression are quite interesting and seem to be related to
mitochondrial adaptation and proliferation. However, the authors should also
present the data for the 1 mM concentration. Moreover, it would be of interest to
determine the expression (or the activity) of the mitochondrial enzyme succinate
dehydrogenase (SDH) in order to determine whether the adaptive up-regulation
of these transcription factors is associated with mitochondrial proliferation. Such
an event has already been observed in muscle of patients suffering from
clevudine-induced myopathy (ref 10). A novel Figure could be added if these new
data are informative.
3- One metabolic consequence of mtDNA depletion and OXPHOS deficiency is
an impairment of fatty acid oxidation, thus leading to lipid accumulation. The
finding that lipid droplets accumulate within treated cells will reinforce the notion
that high concentrations of clevudine have deleterious metabolic consequences
beyond the reduction of ATP content. Lipid accretion could be detected with oil
red O staining or the fluorescent probe Nile red.
4- In this study, clevudine induced a 50-60% reduction in the mtDNA levels. In
most tissues, however, the mtDNA copy number must fall below 20-40% of the
basal levels in order to provoke mitochondrial dysfunction (DiMauro and Schon,
N Engl J Med 2003; Rossignol et al., Biochem J 2003). Moreover, different
investigations showed that nucleoside reverse transcriptase inhibitors such as
stavudine and zidovudine can also induce mitochondrial dysfunction without
significantly depleting mtDNA (Barile et al., Gen Pharmacol 1998; Pan-Zhou et
al., Antimicrob Agents Chemother 2000; Igoudjil et al., Antivir Ther 2007; Igoudjil
et al., Toxicol in Vitro 2008). Thus, the authors cannot exclude the possibility that
clevudine could impair mitochondrial function through mechanisms other than
mtDNA depletion. This concept should be introduced in the discussion.
Other comments (discretionary revisions):
1- More pieces of information should be provided on the INS-1E and HepG2 cell
lines. In particular, the authors should specify that these cell lines correspond to
rat pancreatic beta cells and human hepatoma cells, respectively.
2- The authors should specify that clevudine-induced effects on
glucose-stimulated insulin secretion have been studied after 4 weeks of
treatment.
3- Several articles have shown that uridine is able to prevent NRTI-induced
mitochondrial dysfunction (including mtDNA depletion) in different tissues (see
for instance Lebrecht et al., Hepatology 2007; Lebrecht et al., Arthritis Rheum
2008; Venhoff et al., AIDS 2010). It would be interesting to test whether this
molecule could be able to prevent clevudine-induced mitochondrial dysfunction in
the INS-1E cell line.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests