Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study investigates how lifestyle factors (social isolation and high-fat diet) influence brain sensitivity to circulating IGF-1 and associated mood behaviors. The authors demonstrate that both conditions impair neuronal responses to systemic IGF-1 in the prefrontal cortex while inducing anxiety- and depression-like behaviors. The application of fiber photometry to assess functional brain IGF-1 responsiveness could be a valuable methodological contribution.

The manuscript is clearly written, well-structured, and presents well described evidence for lifestyle-dependent alterations in brain IGF-1 sensitivity. The experimental design is good. Figures are well-designed and effectively communicate the key findings

Minor comments

The manuscript would benefit from additional justification for the IGF-1 administration parameters used in the fiber photometry experiments. Specifically:

1) The IGF-1 dose selection (The 1µg/g body weight requires rationale or supporting literature citation. Was this dose based on previous studies, dose-response preliminary data, or physiological considerations?).

2) The 25-minute post-injection timepoint, what is the rationale for this specific timing? Is this based on known pharmacokinetics of IGF-1 brain entry, or preliminary time-course studies?

In Lines 160-167, the authors conclude that normal pSer³¹⁸-IRS-1 levels indicate intact downstream signaling, leading to the BBB transport hypothesis. This claims could benefit from moderation, presenting the BBB hypothesis as one possibility among several, rather than the logical conclusion, or explain better. 

No more comment, well done. 

 

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

An interesting measure would be the IGF-1 levels in the brain.

Figures need revision. The images are small and of poor quality.

The authors did not present the results related to glucose intolerance and hyperinsulinemia or insulin resistance.

In Figures 2 and 3, what does the 100% baseline represent? Would it not be better to present the data as a percentage relative to the control group?

Some sentences need further clarification and, depending on the response, should be justified. Please explain to the reader what it means that the animals were not randomized. Should they not have been randomized? Additionally, clarify what is meant by "potential confounders were not accounted for."

Please provide more details on how the animals were distributed across the different techniques and tests. Were all animals subjected to both behavioral tests?

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study addresses an important and highly relevant question regarding the influence of lifestyle factors on brain sensitivity to IGF-1. The work is well methodologically performed; however, the reviewer has several questions and comments that require clarification and, potentially, additional data analysis.

Major Remarks:

1. The text and supplementary materials indicate that socially isolated males gained more body weight (Figure S1B), despite exhibiting depression-like behavior and reduced food intake (Figure S1C), which appears contradictory. The authors' further explanation of this phenomenon would be appreciated.
2. The authors note that the high-fat diet (HFD) induced glucose intolerance, hyperinsulinemia, and insulin resistance (citing previous work). However, direct data from intraperitoneal or oral glucose tolerance tests and insulin levels (fasting and/or postprandial) are not presented in the current study. Given that brain insulin resistance is a well-established consequence of HFD and can itself influence behavior [doi: 10.3389/fpsyt.2019.00057; doi: 10.1038/npp.2015.357; doi: 10.1152/physrev.00032.2015] and signaling pathways that overlap with those of IGF-1, including these data would significantly strengthen the manuscript and help partially differentiate the effects of IGF-1 resistance from those of insulin resistance.
3. Adding data on blood insulin and IGF-1 levels in response to IGF-1 and glucose administration would also be valuable, as it could further help disentangle the specific effects of insulin from those of IGF-1.
4. The article presents pooled corticosterone level data for males and females (Fig. 1C). Given the well-documented sex differences in HPA axis stress responses [doi: 10.3390/ijms22063139], it would be informative to present corticosterone levels separately for males and females to rule out a potential confounding effect.
5. To assess the neuronal response to glucose, animals were fasted for 20-24 hours. Such a prolonged fasting period is an extreme stressor for mice, which can significantly alter their metabolic state and welfare. Furthermore, it deviates from standard recommendations (e.g., a 6-hour fast for mice) [doi: 10.1038/s41598-024-72695-3; doi: 10.2147/DMSO.S234665]. A justification for the use of this specific extended fasting protocol is required, along with a discussion of the potential impact of the fasting stressor itself on the obtained results.

Minor Remarks:

1. The quality of the figures is quite low, which made detailed analysis difficult.
2. The figure legends (Figures 1-4) and the Statistics section define the significance levels as *, **, and ***. However, the mark **** also appears on the figures. An explanation for **** (presumably p<0.0001) should be added to the Statistics section or the figure captions.
3. Experimental Design Details:
   a. Mortality: Was any mortality recorded during the 16-week experimental period, particularly following viral transduction? A brief comment in the text would be helpful.
   b. Models duration: Did the animals remain on the HFD and under social isolation for the entire duration of the behavioral testing and final measurements (i.e., the full 18-20 weeks), or was the intervention halted at week 14? This is not entirely clear from the manuscript.

Conclusion: This research is of significant interest and provides an important contribution to our understanding of how lifestyle impacts brain signaling pathways. Addressing the points raised above would help solidify the conclusions of the study and enhance its methodological rigor.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have done a great job revising the manuscript. The experimental design and text are now much clearer. This is a valuable study, and I anticipate their future work will build successfully on these results, overcoming all current limitations.

Minor remarks:

1. Please add an explanation for "****" to the caption of Supplementary Figure 1.
2. Supplementary Figure 3: The unit for corticosterone level appears to be incorrect.
3. Line 87: The "****p<0.0001" should be removed.
4. Figures 2-4: The captions are missing the legend explaining the significance levels.

Author Response

Thank you for your comments.Please see the attachment for reply.

Author Response File: Author Response.pdf