Title: Comparative Analysis of #-Estradiol and Testosterone on Lipid Droplet Accumulation,
and Regulatory Protein Expression in Palmitate/Oleate-Induced Fatty HepG2 Cells
Version: 0 Date: 19 Feb 2025
Reviewer:  Dolores Njoku
Reviewer's report:
In this  study, the authors compared the effects of sex hormones on lipid droplet (LD)
accumulation in palmitate/ oleate-treated HepG2 cells in vitro. The authors utilized MTT method
to determine effective doses of palmitic and oleic acids in HepG2 cells, followed by a combined
dose for inducing LD formation.  The authors evaluated cells in vitro using light microscopy.
The authors measured the effect of beta estradiol and testosterone on lipid droplet formation.
1. My overall assessment of the paper is that the idea is interesting, but I have methodological
concerns that may have skewed the results.
a.  The LD50 does not seem to produce significant differences in measured endpoints.  This
could be because there are no differences or because the dose may still be too high or even
too low.  Extremes of dosing would help clarify whether or not the findings are a dose-related
effect.  Alternatively, the authors could provide guidance regarding a clinically relevant dose of
the saturated and unsaturated fats that would help guide the interpretation.
b. Clinical relevance regarding the doses of estradiol and testosterone would help to comprehend
the result.  In my opinion, they appear high.  Are these the doses that are routinely utilized in this
type of  in vitro study?  As an example, clinical doses of beta estradiol are 30 - 400 picograms/
ml testosterone levels can vary widely.
c. Visually, the oil Red O staining seems equivocal . Suggest photographs that are more
representative of the data analyses that was demonstrated.
2. In the discussion, the following statement was made:  “Although no studies were found
that show the relationship between sex hormones and PPAR#…”   However, a recent study
demonstrated sex differences in hepatic PPARalpha in a mouse model of MASH. (Front Med
(Lausanne). 2024 Aug 20;11:1425528. PMID: 39228797)
3. Minor comment:  In the introductory paragraph, kindly revise “premenopause” to
“premenopausal”.
Title: Comparative Analysis of #-Estradiol and Testosterone on Lipid Droplet Accumulation,
and Regulatory Protein Expression in Palmitate/Oleate-Induced Fatty HepG2 Cells
Version: 0 Date: 19 Feb 2025
Reviewer:  Dolores Njoku
Reviewer's report:
In this  study, the authors compared the effects of sex hormones on lipid droplet (LD)
accumulation in palmitate/ oleate-treated HepG2 cells in vitro. The authors utilized MTT method
to determine effective doses of palmitic and oleic acids in HepG2 cells, followed by a combined
dose for inducing LD formation.  The authors evaluated cells in vitro using light microscopy.
The authors measured the effect of beta estradiol and testosterone on lipid droplet formation.
1. My overall assessment of the paper is that the idea is interesting, but I have methodological
concerns that may have skewed the results.
a.  The LD50 does not seem to produce significant differences in measured endpoints.  This
could be because there are no differences or because the dose may still be too high or even
too low.  Extremes of dosing would help clarify whether or not the findings are a dose-related
effect.  Alternatively, the authors could provide guidance regarding a clinically relevant dose of
the saturated and unsaturated fats that would help guide the interpretation.
b. Clinical relevance regarding the doses of estradiol and testosterone would help to comprehend
the result.  In my opinion, they appear high.  Are these the doses that are routinely utilized in this
type of  in vitro study?  As an example, clinical doses of beta estradiol are 30 - 400 picograms/
ml testosterone levels can vary widely.
c. Visually, the oil Red O staining seems equivocal . Suggest photographs that are more
representative of the data analyses that was demonstrated.
2. In the discussion, the following statement was made:  “Although no studies were found
that show the relationship between sex hormones and PPAR#…”   However, a recent study
demonstrated sex differences in hepatic PPARalpha in a mouse model of MASH. (Front Med
(Lausanne). 2024 Aug 20;11:1425528. PMID: 39228797)
3. Minor comment:  In the introductory paragraph, kindly revise “premenopause” to
“premenopausal”.
Please review this paper.
user
Title: Comparative Analysis of #-Estradiol and Testosterone on Lipid Droplet Accumulation,
and Regulatory Protein Expression in Palmitate/Oleate-Induced Fatty HepG2 Cells
Version: 0 Date: 19 Feb 2025
Reviewer:  Dolores Njoku
Reviewer's report:
In this  study, the authors compared the effects of sex hormones on lipid droplet (LD)
accumulation in palmitate/ oleate-treated HepG2 cells in vitro. The authors utilized MTT method
to determine effective doses of palmitic and oleic acids in HepG2 cells, followed by a combined
dose for inducing LD formation.  The authors evaluated cells in vitro using light microscopy.
The authors measured the effect of beta estradiol and testosterone on lipid droplet formation.
1. My overall assessment of the paper is that the idea is interesting, but I have methodological
concerns that may have skewed the results.
a.  The LD50 does not seem to produce significant differences in measured endpoints.  This
could be because there are no differences or because the dose may still be too high or even
too low.  Extremes of dosing would help clarify whether or not the findings are a dose-related
effect.  Alternatively, the authors could provide guidance regarding a clinically relevant dose of
the saturated and unsaturated fats that would help guide the interpretation.
b. Clinical relevance regarding the doses of estradiol and testosterone would help to comprehend
the result.  In my opinion, they appear high.  Are these the doses that are routinely utilized in this
type of  in vitro study?  As an example, clinical doses of beta estradiol are 30 - 400 picograms/
ml testosterone levels can vary widely.
c. Visually, the oil Red O staining seems equivocal . Suggest photographs that are more
representative of the data analyses that was demonstrated.
2. In the discussion, the following statement was made:  “Although no studies were found
that show the relationship between sex hormones and PPAR#…”   However, a recent study
demonstrated sex differences in hepatic PPARalpha in a mouse model of MASH. (Front Med
(Lausanne). 2024 Aug 20;11:1425528. PMID: 39228797)
3. Minor comment:  In the introductory paragraph, kindly revise “premenopause” to
“premenopausal”.
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