Effects of Chronic Elevation in Plasma Membrane Cholesterol on the Function of Human Na⁺/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Jessica Y. Idowu et al. is interesting and well presented.

Some suggestions:
1. Could you provide complete information on the antibodies used, including brand, catalog, country, etc.? Thank you.
2. Given the type of study, are you considering using ANCOVA as a statistic?
3. Could you please add conclusions and perspectives? Thank you.
4. Are there any limitations to your study?
5. The protocol, because the research committee was approved, could you provide the name, registration number, and approval date? Thank you.

Author Response

We would like to thank all reviewers for their constructive comments and suggestions. We have incorporated or addressed all these comments, and this resulted in a much-improved manuscript. In the following, we provide a point-by-point response to the individual comments and suggestions in italics.

Comment 1. Could you provide complete information on the antibodies used, including brand, catalog, country, etc.? Thank you.

Response 1. We apologize if this was not absolutely clear, but this information is already included in the first paragraph of the Materials and Methods section; therefore, we decided not to change anything.

Comment 2. Given the type of study, are you considering using ANCOVA as a statistic?

Response 2. We have analyzed the data using ANOVA followed by "Dunnett's multiple comparisons test". To the best of our knowledge this is an appropriate statistical analysis of the presented data.

Comment 3. Could you please add conclusions and perspectives? Thank you.

Response 3. Conclusions and Perspectives are included in the last paragraph.

 
Comment 4. Are there any limitations to your study?
Response 4. Yes, there are limitations to our study, and we mentioned these limitations in the submitted manuscript in lines 276 – 293.

 

Comment 5. The protocol, because the research committee was approved, could you provide the name, registration number, and approval date? Thank you.

Response 5. We are unsure what the reviewer asks here because we did not perform any trials or animal experiments that would have required a registration number.

Reviewer 2 Report

Comments and Suggestions for Authors

I read with interest the manuscript “Effects of Chronic Elevation in Plasma Membrane Cholesterol on the Function of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)”.

This manuscript addresses a highly relevant topic: how chronic changes in plasma membrane cholesterol levels affect key hepatic transporters, NTCP and OCT1. The work builds upon previous studies that examined acute cholesterol effects and aims to mimic the more clinically relevant chronic conditions seen in obesity-related liver disease. The experimental design is rigorous, and the results are clearly presented and well contextualized within existing literature. However, some limitations remain that should be acknowledged or addressed.

Major concern

1. The use of HEK293 cells stably expressing NTCP and OCT1 is a reasonable approach for transporter studies, but the authors acknowledge that this model lacks hepatocyte-specific features. While future plans to develop HepG2 models are mentioned, the current manuscript would benefit from a more thorough discussion of how this limitation affects data interpretation and translatability to liver physiology.
2. The 3-fold increase in OCT1 transport capacity with chronic cholesterol exposure is a key finding, particularly in relation to metformin pharmacokinetics. However, the claim that "patients might benefit from dose adjustments" needs to be tempered. In vivo confirmation is needed before proposing changes to therapeutic regimens.
3. The discussion on CRAC motifs and potential direct interactions between cholesterol and OCT1 is compelling. However, as this is speculative and not experimentally demonstrated here, the authors should present these ideas more cautiously and clearly distinguish them from findings.
4. While the addition of ACAT inhibitors provided further insights into the impact of unesterified cholesterol, the potential confounding direct effects of these compounds on transporter function are not sufficiently resolved. The preliminary data on NTCP inhibition are noted but need either experimental confirmation or a clearer acknowledgment of the uncertainty.

Minor comments:

1. Figures are generally well-designed. However, including representative blots alongside quantified graphs (e.g., in Figure 3) is good practice, and care should be taken to ensure band intensity correlates with the quantification.
2. The terms “capacity” and “turnover number” are used somewhat interchangeably with Vmax. Consider clarifying that the increase in Vmax post-normalization reflects an increased intrinsic transport activity per surface-expressed transporter.
3. Define abbreviations like ACAT and MASLD/MASH clearly at first mention.

Author Response

We would like to thank all reviewers for their constructive comments and suggestions. We have incorporated or addressed all these comments, and this resulted in a much-improved manuscript. In the following, we provide a point-by-point response to the individual comments and suggestions in italics.

Reviewer 2:

I read with interest the manuscript “Effects of Chronic Elevation in Plasma Membrane Cholesterol on the Function of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)”.

This manuscript addresses a highly relevant topic: how chronic changes in plasma membrane cholesterol levels affect key hepatic transporters, NTCP and OCT1. The work builds upon previous studies that examined acute cholesterol effects and aims to mimic the more clinically relevant chronic conditions seen in obesity-related liver disease. The experimental design is rigorous, and the results are clearly presented and well contextualized within existing literature. However, some limitations remain that should be acknowledged or addressed.

 

We would like to thank this reviewer for the positive feedback.

Major concern

Comment 1. The use of HEK293 cells stably expressing NTCP and OCT1 is a reasonable approach for transporter studies, but the authors acknowledge that this model lacks hepatocyte-specific features. While future plans to develop HepG2 models are mentioned, the current manuscript would benefit from a more thorough discussion of how this limitation affects data interpretation and translatability to liver physiology.

Response 1. We have expanded the discussion with the following paragraph “While HEK293 cells are a great tool to express individual transporters and manipulate growth conditions, they have several disadvantages compared to human hepatocytes. First, they only express a limited number of hepatocellular transporters, normally only one transporter at a time, and frequently this transporter is overexpressed compared to hepatocytes. This prevents potential effects from the interactions of multiple transporters that are expressed in the same hepatocyte. Second, HEK293 cells to not express all the metabolizing enzymes that human hepatocytes express. Consequently, especially under the more chronic conditions used in this study, potential effects of such liver enzymes are missing in HEK293 cells. And third, compared to the situation in the liver where hepatocytes interact with Kupffer cells, stellate cells and potentially other cell types, HEK293 cells in culture cannot replicate these more complex interactions.”

 

Comment 2. The 3-fold increase in OCT1 transport capacity with chronic cholesterol exposure is a key finding, particularly in relation to metformin pharmacokinetics. However, the claim that "patients might benefit from dose adjustments" needs to be tempered. In vivo confirmation is needed before proposing changes to therapeutic regimens.

Response 2. We agree with the reviewer, and we have replaced the statement in the abstract by “might affect drug therapy”; in the conclusion section we change the statement to “drug therapy in patients treated with OCT1-transported drugs might be affected”. We hope that the reviewer agrees that these statements are less strong and imply further studies in human subjects.

 

Comment 3. The discussion on CRAC motifs and potential direct interactions between cholesterol and OCT1 is compelling. However, as this is speculative and not experimentally demonstrated here, the authors should present these ideas more cautiously and clearly distinguish them from findings.

Response 3. To clarify that we did not yet investigate this experimentally, we have modified the discussion as follows: “Another possibility is that cholesterol interacts directly with the transport proteins, and we will investigate this possibility in future experiments.” And: “Therefore, in future experiments we will investigate whether”. We hope that these changes more clearly indicate that this is currently completely speculative and not supported by any experiments.

 

Comment 4. While the addition of ACAT inhibitors provided further insights into the impact of unesterified cholesterol, the potential confounding direct effects of these compounds on transporter function are not sufficiently resolved. The preliminary data on NTCP inhibition are noted but need either experimental confirmation or a clearer acknowledgment of the uncertainty.

Response 4. We have added the results of experiments with OCT1 (n=3) and NTCP (n=2) in Figure S1 in the supplementary material.

 

Minor comment 1. Figures are generally well-designed. However, including representative blots alongside quantified graphs (e.g., in Figure 3) is good practice, and care should be taken to ensure band intensity correlates with the quantification.

Response to minor comment 1. All blots that were used for the quantification in Figure 3 are included in the supplementary material.

 

Minor comment 2. The terms “capacity” and “turnover number” are used somewhat interchangeably with Vmax. Consider clarifying that the increase in Vmax post-normalization reflects an increased intrinsic transport activity per surface-expressed transporter.

Response to minor comment 2. We have modified the statement in the discussion to reflect this, and it now reads as follows:” This result was mainly driven by an increase in Vmax which reflects an increased intrinsic transport activity per surface-expressed transporter since the results were normalized for protein at the plasma membrane.”

 

Minor comment 3. Define abbreviations like ACAT and MASLD/MASH clearly at first mention.

Response to minor comment 3. We tried to make sure all the abbreviations are defined at the first mention.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review the article entitled "Effects of Chronic Elevation in Plasma Membrane Cholesterol on the Function of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)".

This study offers pertinent data that could have substantial clinical implications, particularly in the management of individuals with MASLD/MASH. An suitable approach was employed, encompassing stable HEK293 cell line cultures, biochemical assays, surface biotinylation assays, among others, with relevant prior work duly cited.

The authors appropriately highlight the limitations of the study, demonstrating significant research maturity.

I have no objections to the current version of the manuscript; nevertheless, I recommend that the authors elaborate on the study's limitations and explore potential solutions to this issue.

I congratulate the authors on their efforts and excellent work!

Author Response

We would like to thank all reviewers for their constructive comments and suggestions. We have incorporated or addressed all these comments, and this resulted in a much-improved manuscript. In the following, we provide a point-by-point response to the individual comments and suggestions in italics.

Thank you for the opportunity to review the article entitled "Effects of Chronic Elevation in Plasma Membrane Cholesterol on the Function of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)".

This study offers pertinent data that could have substantial clinical implications, particularly in the management of individuals with MASLD/MASH. An suitable approach was employed, encompassing stable HEK293 cell line cultures, biochemical assays, surface biotinylation assays, among others, with relevant prior work duly cited.

The authors appropriately highlight the limitations of the study, demonstrating significant research maturity.

I have no objections to the current version of the manuscript; nevertheless, I recommend that the authors elaborate on the study's limitations and explore potential solutions to this issue.

I congratulate the authors on their efforts and excellent work!

 

Response: We have expanded the discussion with the following paragraph “While HEK293 cells are a great tool to express individual transporters and manipulate growth conditions, they have several disadvantages compared to human hepatocytes. First, they only express a limited number of hepatocellular transporters, normally only one transporter at a time, and frequently this transporter is overexpressed compared to hepatocytes. This prevents potential effects from the interactions of multiple transporters that are expressed in the same hepatocyte. Second, HEK293 cells to not express all the metabolizing enzymes that human hepatocytes express. Consequently, especially under the more chronic conditions used in this study, potential effects of such liver enzymes are missing in HEK293 cells. And third, compared to the situation in the liver where hepatocytes interact with Kupffer cells, stellate cells and potentially other cell types, HEK293 cells in culture cannot replicate these more complex interactions.”

Reviewer 4 Report

Comments and Suggestions for Authors

The authors aim to analyze the indirect causal relationship between increased cholesterol levels and the protein expression of two hepatocyte membrane transporters in cell cultures.

Some issues undermine the quality of the work:

 

1. The authors should modify or clearly explain when referring to "free cholesterol in the plasma membrane," as this term is confusing throughout the manuscript. Usually, the term "free cholesterol" refers to cholesterol in plasma or blood, not cholesterol contained within the membrane where cholesterol is not free.
2. The authors should justify why incubation with cholesterol for 30 minutes mimics obese patients.

 

3. The causal relationship that led the authors to study NTCP and OCT1 in hepatocytes and cholesterol should be justified.

 

4. Line 18. "Cholesterol contained in cells" refers to cholesterol in the plasma membrane or in cytoplasmic organelles?

 

5. Line 25. Increase or decrease? Specify which.
6. The abstract should be improved to summarize the manuscript. As currently expressed, it raises too many questions. The receptor substrates should be specified.
7. Line 32. "Free cholesterol." This term refers to free cholesterol in the blood.

 

8. The introduction should be expanded to explain the causal relationship between the cholesterol study and the two types of transporters.
9. Line 45. Free cholesterol in the plasma membrane?
10. Water-soluble cholesterol? This term should be explained. What chemical form does it have? Could it have implications in experiments by preventing the inclusion of this type of cholesterol in the plasma membrane?

 

11. 2.5. The significance level used in the study should be indicated.

 

12. Lines 95-97. Indicate the reference of the statement.

 

13. Line 100. 107, 117, 135. Free cholesterol levels???
14. Line 110: 0.8 mM of ??? 0.4 mM of ???
15. Fig. 1. Free cholesterol in the graph refers to membrane cholesterol or free cholesterol. Indicate the cholesterol concentration used on the graph. Indicate what this means (% control).
16. Fig. 2. Uptake of ….?
17. Line 137. Plasma membrane cholesterol is free cholesterol.
18. Indicate “surface biotinylation” and explain.
19. Why do the authors use Na/K ATPase as a loading control?
20. Fig. 3. Clearly indicate “Surface expression” of…. on all axes of the graphs and the concentrations used. The figures should be self-explanatory.
21. Fig. 3C-D. The decrease in both transporters relative to the loading control is not clearly visible, as it decreases proportionally.
22. Table 1. Line 192. Average = mean? SEM and SD should be used consistently in the manuscript.
23. Table 1. Indicate the comparison made at the significant value.
24. Explain how the use of ACAT inhibitors affects the decrease in membrane activity when using a modified cholesterol with greater solubility.
25. Line 247. This could be demonstrated by colocalization.
26. Line 248. References are missing.
27. Line 256. Loading the plasma membrane?
28. Line 273. Our studies? Where are the references?

Author Response

We would like to thank all reviewers for their constructive comments and suggestions. We have incorporated or addressed all these comments, and this resulted in a much-improved manuscript. In the following, we provide a point-by-point response to the individual comments and suggestions in italics.

The authors aim to analyze the indirect causal relationship between increased cholesterol levels and the protein expression of two hepatocyte membrane transporters in cell cultures.

Some issues undermine the quality of the work:

We thank the reviewer for this extensive and detailed comments. We have responded to all the comments and made most of the suggested changes.

 

Comment 1. The authors should modify or clearly explain when referring to "free cholesterol in the plasma membrane," as this term is confusing throughout the manuscript. Usually, the term "free cholesterol" refers to cholesterol in plasma or blood, not cholesterol contained within the membrane where cholesterol is not free.

Response 1. We thank the reviewer for asking for this clarification. We are using the term “free cholesterol” to mean cholesterol that is not attached to a fatty acid. This is also known as unesterified cholesterol and plays an important role in the plasma membrane. We have added a sentence to clarify this as the second sentence of the introduction: “By free cholesterol we mean cholesterol that is not attached to a fatty acid and thus unesterified.”

 

Comment 2. The authors should justify why incubation with cholesterol for 30 minutes mimics obese patients.

Response 2. We apologize for this misunderstanding. We never indicated that a 30-minute incubation with cholesterol mimics obese patients. Based on previous findings by others, it is known that the unesterified or free cholesterol content in the liver of obese subjects is increased as compared to normal controls (PMID: 28097795 and PMID: 17654743). In the presented study where we used a more chronic condition, we wanted to mimic what would be seen in obese patients.

 

Comment 3. The causal relationship that led the authors to study NTCP and OCT1 in hepatocytes and cholesterol should be justified.

Response 3. The introduction mentions that this study was a follow-up study of a previously published study where we investigated the acute effect of a 30-minute incubation with free cholesterol on the expression and function of NTCP and OCT1.

 

Comment 4. Line 18. "Cholesterol contained in cells" refers to cholesterol in the plasma membrane or in cytoplasmic organelles?

Response 4. It refers to cholesterol in the plasma membrane because we measured free (unesterified) cholesterol. Free cholesterol is mainly in the plasma membrane while cholesterol in lipid droplets is mainly esterified.

 

Comment 5. Line 25. Increase or decrease? Specify which.

Response 5. Both, increased for OCT1 and decreased for NTCP.

 

Comment 6. The abstract should be improved to summarize the manuscript. As currently expressed, it raises too many questions. The receptor substrates should be specified.

Response 6. We apologize if the abstract was not clear to this reviewer. None of the other reviewers complained about the clarity of the abstract, so we respectfully decline to change it. Furthermore, we do not know what this reviewer means by receptor substrates should be specified. If this reviewer means the transported substrates they are clearly given in the abstract. “determined their function by measuring uptake of radioactive taurocholate or MPP⁺.”

 

Comment 7. Line 32. "Free cholesterol." This term refers to free cholesterol in the blood.

Response 7. Please see response 1.

 

Comment 8. The introduction should be expanded to explain the causal relationship between the cholesterol study and the two types of transporters.

Response 8. The introduction mentions that this study was a follow-up study of a previously published study where we investigated the acute effect of a 30-minute incubation with free cholesterol on the expression and function of NTCP and OCT1.

 

Comment 9. Line 45. Free cholesterol in the plasma membrane?

Response 9. Please see response 1.

 

Comment 10. Water-soluble cholesterol? This term should be explained. What chemical form does it have? Could it have implications in experiments by preventing the inclusion of this type of cholesterol in the plasma membrane?

Response 10. Water-soluble cholesterol is cholesterol that is balanced with methyl-β-cyclodextrin and is sold by Sigma. We have added this specification in the materials and methods section. 

 

Comment 11. 2.5. The significance level used in the study should be indicated.

Response 11. We mention that “Significance and p-values are given in the figure legends” because we have calculated different significance levels.

 

Comment 12. Lines 95-97. Indicate the reference of the statement.

Response 12. There is no reference for this statement because we established this in this study. Thus, it cannot be referenced.

 

Comment 13. Line 100. 107, 117, 135. Free cholesterol levels???

Response 13. We apologize but we do not understand what this reviewer means by this comment. We measured increased levels of free (unesterified) cholesterol in these cells.

 

Comment 14. Line 110: 0.8 mM of ??? 0.4 mM of ???

Response 14. We apologize if this statement was unclear. We have modified it to read “with much higher concentrations of free cholesterol (0.8mM for NTCP and 0.4 mM for OCT1)”

 

Comment 15. Fig. 1. Free cholesterol in the graph refers to membrane cholesterol or free cholesterol. Indicate the cholesterol concentration used on the graph. Indicate what this means (% control).

Response 15. Please see response 1.

 

Comment 16. Fig. 2. Uptake of ….?

Response 16. Taurocholate and MPP⁺ “Uptake of (A) (100 μM) [³H]-taurocholate for NTCP- and (B) (16.7 nM) [³H]-MPP⁺”

 

Comment 17. Line 137. Plasma membrane cholesterol is free cholesterol.

Response 17. We have added “free” in this line.

 

Comment 18. Indicate “surface biotinylation” and explain.

Response 18. We are sorry but we do not understand what this reviewer means here. We refer to the detailed methods published in our previous study in order not to repeat methods. If this is absolutely necessary, we can add more details in the materials section, but we would prefer to just refer to the previously published method.

 

Comment 19. Why do the authors use Na/K ATPase as a loading control?

Response 19. We have used Na⁺/K⁺-ATPase as a loading control because Na⁺/K⁺-ATPase is intrinsically expressed at the plasma membrane of most cells, including HEK293 cells.

 

 

Comment 20. Fig. 3. Clearly indicate “Surface expression” of…. on all axes of the graphs and the concentrations used. The figures should be self-explanatory.

Response 20. We have changed the y-axes in Figure 6 to what they are in Figure 3. We agree that figures with their legends should be self-explanatory.

 

Comment 21. Fig. 3C-D. The decrease in both transporters relative to the loading control is not clearly visible, as it decreases proportionally.

Response 21. We are not absolutely clear about this comment. However, we have included all the blots used to quantitate the western blots in the supplemental material, and we hope that this addition clarifies our finding/calculation.

 

Comment 22. Table 1. Line 192. Average = mean? SEM and SD should be used consistently in the manuscript.

Response 22. We have changed average to mean. We present SEM in the Table for the kinetics because GraphPad Prism only calculates SEM for the Km and the Vmax values, but not SD.

 

Comment 23. Table 1. Indicate the comparison made at the significant value.

Response 23. We have added “as compared to the control condition”

 

Comment 24. Explain how the use of ACAT inhibitors affects the decrease in membrane activity when using a modified cholesterol with greater solubility.

Response 24. We are unsure what the reviewer means here. We did not measure membrane activity, and we don’t know what modified cholesterol would have greater solubility.

 

Comment 25. Line 247. This could be demonstrated by colocalization.

Response 25. We are unsure how this would work given that the plasma membrane contains free cholesterol that most likely does not interact with the two transporters. We could envision that perhaps cryo-EM could be able to resolve this but techniques that are available to us like antibodies and confocal imaging are not able to demonstrate this colocalization.

 

Comment 26. Line 248. References are missing.

Response 26. Reference 32 and 33 are listed in line 252.

 

Comment 27. Line 256. Loading the plasma membrane?

Response 27. Increasing the amount of free (unesterified) cholesterol.

 

Comment 28. Line 273. Our studies? Where are the references?

Response 28. We changed the wording to “The presented studies”, to make clear that we refer to the limitations of the studies that we just presented.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

satisfied

Reviewer 4 Report

Comments and Suggestions for Authors

All required aspects have been properly reviewed by the authors.