Reviewer's report
Title:High-dose clevudine impairs mitochondrial function and glucose-stimulated
insulin secretion in INS-1E cells
Version: 1Date:25 August 2011
Reviewer: Nazzareno Capitanio
Reviewer's report:
The study by Y-O Jang and co-workers provides evidence that the NRTI
clevudine induces, at relatively high concentrations, impairment of mitochondrial
functions in INS-1E and HepG2 cells. In particular, as expected, clevudine
treatment causes a significant reduction of the mtDNA copy number/cell likely
due to inhibition of the mitochondrial DNA polgamma. Moreover, in INS-1E cells
the drug causes a marked decrease of insulin release upon high
glucose-stimulation. The authors link this defective insulin release to alteration of
the ATP level in clevudine-treated cells.
All these effects are attained at non-pharmacological concentrations of the drug
therefore they are of limited clinical relevance as correctly stated by the authors.
However, individual differences in the genetic background of the DNA polgamma
may render some subjects more susceptible to the mitotoxicity of the drug
thereby accounting for the limited number of cases of miopathy and diabetes
reported as side-effect in patients long-treated with clevudine.
The manuscript is well written, the data clearly presented and the results of
interest. However, the following specific points/concerns are raised and required
to be satisfied to make the paper acceptable for publication.
Major points
1. The reported decrease in the Cox-1 mRNA (Fig. 1B,D) does not necessarily
mean a decrease of the protein and therefore of the related function.
Measurement of the activity of the cytochrome c oxidase on cell lysate may help
to strengthen the authors’ conclusions.
2. The MTT assay is extremely vague to measure the mitochondrial enzymatic
activity. A more specific assay like measurement of the citrate synthase activity is
suggested (Barrientos A. Methods 2002;26:307-316). If the authors had the
possibility to measure the oxygen consumption rates in intact cells by
respirometry this would very clearly make the point on the oxidative
phosphorylation capacity.
3. The cause-effect relationship between the decreased ATP level and the
impaired insulin release does not appear very straightforward. Comparing Figs.
2B and 3 it seems that some inhibition of insulin release is observable already at
100 microM clevudine (high-glucose setting) which however did not change the
ATP level. The authors are asked to clarify this point. If the release of insulin by
INS-1E cells is sensitive to the mitochondrial ATP, oligomycin-treatment in
control cells should mimic the observed clevudine-mediated decrease of insulin
release therefore proving the cause-effect relationship.
Minor points
4. The authors should clarify the reason of the long 4 weeks-treatment with
clevudine. A time course of the reported effects has been established? Is there
some effect on the cell growth?
5. Fig. 1A. The normalization of the mtDNA should be better defined in Materials
and Methods. In the same Figure the significance (P value) of the differences
should be included (same for Fig. 2C).
6. In the MTT assay the absorbance should have been normalized to the cell
number per well. If the number of cells/well is the same why the authors state (in
the Results Section) that the observed decrease implies a reduction in cells
number.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:High-dose clevudine impairs mitochondrial function and glucose-stimulated
insulin secretion in INS-1E cells
Version: 1Date:25 August 2011
Reviewer: Nazzareno Capitanio
Reviewer's report:
The study by Y-O Jang and co-workers provides evidence that the NRTI
clevudine induces, at relatively high concentrations, impairment of mitochondrial
functions in INS-1E and HepG2 cells. In particular, as expected, clevudine
treatment causes a significant reduction of the mtDNA copy number/cell likely
due to inhibition of the mitochondrial DNA polgamma. Moreover, in INS-1E cells
the drug causes a marked decrease of insulin release upon high
glucose-stimulation. The authors link this defective insulin release to alteration of
the ATP level in clevudine-treated cells.
All these effects are attained at non-pharmacological concentrations of the drug
therefore they are of limited clinical relevance as correctly stated by the authors.
However, individual differences in the genetic background of the DNA polgamma
may render some subjects more susceptible to the mitotoxicity of the drug
thereby accounting for the limited number of cases of miopathy and diabetes
reported as side-effect in patients long-treated with clevudine.
The manuscript is well written, the data clearly presented and the results of
interest. However, the following specific points/concerns are raised and required
to be satisfied to make the paper acceptable for publication.
Major points
1. The reported decrease in the Cox-1 mRNA (Fig. 1B,D) does not necessarily
mean a decrease of the protein and therefore of the related function.
Measurement of the activity of the cytochrome c oxidase on cell lysate may help
to strengthen the authors’ conclusions.
2. The MTT assay is extremely vague to measure the mitochondrial enzymatic
activity. A more specific assay like measurement of the citrate synthase activity is
suggested (Barrientos A. Methods 2002;26:307-316). If the authors had the
possibility to measure the oxygen consumption rates in intact cells by
respirometry this would very clearly make the point on the oxidative
phosphorylation capacity.
3. The cause-effect relationship between the decreased ATP level and the
impaired insulin release does not appear very straightforward. Comparing Figs.
2B and 3 it seems that some inhibition of insulin release is observable already at
100 microM clevudine (high-glucose setting) which however did not change the
ATP level. The authors are asked to clarify this point. If the release of insulin by
INS-1E cells is sensitive to the mitochondrial ATP, oligomycin-treatment in
control cells should mimic the observed clevudine-mediated decrease of insulin
release therefore proving the cause-effect relationship.
Minor points
4. The authors should clarify the reason of the long 4 weeks-treatment with
clevudine. A time course of the reported effects has been established? Is there
some effect on the cell growth?
5. Fig. 1A. The normalization of the mtDNA should be better defined in Materials
and Methods. In the same Figure the significance (P value) of the differences
should be included (same for Fig. 2C).
6. In the MTT assay the absorbance should have been normalized to the cell
number per well. If the number of cells/well is the same why the authors state (in
the Results Section) that the observed decrease implies a reduction in cells
number.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:High-dose clevudine impairs mitochondrial function and glucose-stimulated
insulin secretion in INS-1E cells
Version: 1Date:25 August 2011
Reviewer: Nazzareno Capitanio
Reviewer's report:
The study by Y-O Jang and co-workers provides evidence that the NRTI
clevudine induces, at relatively high concentrations, impairment of mitochondrial
functions in INS-1E and HepG2 cells. In particular, as expected, clevudine
treatment causes a significant reduction of the mtDNA copy number/cell likely
due to inhibition of the mitochondrial DNA polgamma. Moreover, in INS-1E cells
the drug causes a marked decrease of insulin release upon high
glucose-stimulation. The authors link this defective insulin release to alteration of
the ATP level in clevudine-treated cells.
All these effects are attained at non-pharmacological concentrations of the drug
therefore they are of limited clinical relevance as correctly stated by the authors.
However, individual differences in the genetic background of the DNA polgamma
may render some subjects more susceptible to the mitotoxicity of the drug
thereby accounting for the limited number of cases of miopathy and diabetes
reported as side-effect in patients long-treated with clevudine.
The manuscript is well written, the data clearly presented and the results of
interest. However, the following specific points/concerns are raised and required
to be satisfied to make the paper acceptable for publication.
Major points
1. The reported decrease in the Cox-1 mRNA (Fig. 1B,D) does not necessarily
mean a decrease of the protein and therefore of the related function.
Measurement of the activity of the cytochrome c oxidase on cell lysate may help
to strengthen the authors’ conclusions.
2. The MTT assay is extremely vague to measure the mitochondrial enzymatic
activity. A more specific assay like measurement of the citrate synthase activity is
suggested (Barrientos A. Methods 2002;26:307-316). If the authors had the
possibility to measure the oxygen consumption rates in intact cells by
respirometry this would very clearly make the point on the oxidative
phosphorylation capacity.
3. The cause-effect relationship between the decreased ATP level and the
impaired insulin release does not appear very straightforward. Comparing Figs.
2B and 3 it seems that some inhibition of insulin release is observable already at
100 microM clevudine (high-glucose setting) which however did not change the
ATP level. The authors are asked to clarify this point. If the release of insulin by
INS-1E cells is sensitive to the mitochondrial ATP, oligomycin-treatment in
control cells should mimic the observed clevudine-mediated decrease of insulin
release therefore proving the cause-effect relationship.
Minor points
4. The authors should clarify the reason of the long 4 weeks-treatment with
clevudine. A time course of the reported effects has been established? Is there
some effect on the cell growth?
5. Fig. 1A. The normalization of the mtDNA should be better defined in Materials
and Methods. In the same Figure the significance (P value) of the differences
should be included (same for Fig. 2C).
6. In the MTT assay the absorbance should have been normalized to the cell
number per well. If the number of cells/well is the same why the authors state (in
the Results Section) that the observed decrease implies a reduction in cells
number.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
assistant