Non-Pharmaceutical Interventions Based on Diet Restriction and Exercise Improve Morphology and Function of Fatty Pancreas in Male WBN/Kob-Lepr (Fa/Fa) Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work is well performed and clearly presented.  The conclusion: “These results demonstrate that cellular and subcellular fatty deterioration is not inevitably linked to inflammation, and exercise effectively improves fatty pancreas deterioration by skeletal muscle activity linked through the circulatory network.” It is interesting and clinically important. 

 

The authors described the advantage of using the WKF rat strain was developed by introducing a leptin deficiency gene into the original WBN/Kob (lean) rat genome, which possesses a vulnerable pancreas that spontaneously develops chronic pancreatitis and secondary diabetes.  Similar mouse models (such as db/db mouse) exist, and numerous relevant studies have been conducted with the mouse models.  Please discuss the relevant studies in db/db mouse and point out the advantages of using the WKF rat over db/db mouse (there are several).  This will strengthen the manuscript and validate the WKF rat as an existing alternative. 

 

Author Response

Reviewer 1

 

This work is well performed and clearly presented.  The conclusion: “These results demonstrate that cellular and subcellular fatty deterioration is not inevitably linked to inflammation, and exercise effectively improves fatty pancreas deterioration by skeletal muscle activity linked through the circulatory network.” It is interesting and clinically important.

 

The authors described the advantage of using the WKF rat strain was developed by introducing a leptin deficiency gene into the original WBN/Kob (lean) rat genome, which possesses a vulnerable pancreas that spontaneously develops chronic pancreatitis and secondary diabetes.  Similar mouse models (such as db/db mouse) exist, and numerous relevant studies have been conducted with the mouse models.  Please discuss the relevant studies in db/db mouse and point out the advantages of using the WKF rat over db/db mouse (there are several).  This will strengthen the manuscript and validate the WKF rat as an existing alternative.

 

Thank you for your interest in the possibility that fatty degeneration may not be associated with inflammation. Reviewer 2 highlighted that the evidence presented in our study is insufficient to support such a definitive conclusion; therefore, we have moderated the wording in the revised manuscript. In addition, Reviewer 2 noted that our statements regarding “skeletal muscle–pancreas crosstalk” and the extrapolation to human pathophysiology were overly speculative. In response, we removed Figure 6 and revised these sections express our views more cautiously.　 Discussion (previous version of the manuscript) pvL311–341　In addition, we have expanded the discussion in line with the comments provided by Reviewer 2.

 

Also, thank you very much for your valuable comments.

In accordance with your suggestions, we have added a description of the characteristics of the obese mouse model (db/db mouse) based on previous studies, and included a comparison with the WKF rat.  Introduction L82–86, Discussion L317–320

Reviewer 2 Report

Comments and Suggestions for Authors

 

This manuscript investigates the effects of diet restriction and diet restriction combined with exercise on fatty pancreas morphology and function in WBN/Kob-Lepr fa/fa rats. The authors conclude that fatty deterioration is not inherently pro-inflammatory and that exercise is required to normalize serum FFA levels and restore subcellular architecture.

However, several conceptual, methodological, and interpretational limitations need to be addressed before the manuscript can be considered for publication.

• The study lacks an exercise-only group, preventing clear discrimination between the independent effects of exercise and the interaction effects of exercise combined with diet restriction.
• Only male rats were included. Given that diabetes and metabolic dysfunction are sex-dependent conditions, inclusion of female animals would strengthen the study and improve generalizability.
• The 6-week intervention may not adequately capture long-term progression of fatty pancreas and inflammation.
• Conclusions regarding implications for human metabolic dysfunction-associated fatty pancreas disease may be overstated, given the specific genetic background of the animal model used.
• The manuscript does not clearly describe how the pancreas was harvested and whether a standardized method was used to ensure consistent tissue collection for weight measurements.
• The manuscript reports decreased pancreatic protein content in obese rats without providing mechanistic explanation or supporting references.
• Inflammation, ER stress, and autophagy are complex processes involving multiple signaling pathways. Assessment of only a single marker for each process is insufficient to conclude that these pathways were comprehensively evaluated.
• The manuscript does not adequately explain the roles of LC3-I or LC3-II in autophagy and interpret the findings accordingly.
• The term "Pancreatic B cells" is potentially misleading, as B cells commonly refer to immune cells. The manuscript should clarify whether pancreatic beta cells are intended and revise terminology accordingly.
• Only serum FFA and triglycerides were measured. No detailed assessment of pancreatic lipid species or lipotoxic intermediates was performed.
• Electron microscopy findings are descriptive without morphometric quantification of mitochondrial damage, ER dilation, or lipid droplet accumulation.
• The designation of "early pancreatitis" lacks standardized pathological scoring and fibrosis evaluation.
• Effects attributed to diet restriction or exercise may partly reflect differences in body weight and metabolic load that were not fully controlled.
• The units used for glucose and insulin in the HOMA-IR formula appear inconsistent with standard methodology, raising concerns about the validity of insulin resistance assessment.
• The manuscript does not specify how western blot data were normalized for loading (e.g., housekeeping protein, total protein staining).
• Original, uncropped western blot images are not included, limiting assessment of data integrity.
• The manuscript does not clearly state whether allocation, outcome assessment, or histological analyses were performed in a blinded and randomized manner.
• The conclusion that fatty deterioration is not inherently pro-inflammatory is not sufficiently supported by the limited inflammatory assessment and single time point analysis.
• Claims regarding skeletal muscle-pancreas crosstalk and myokine involvement are speculative and not directly supported by experimental data.
• The discussion does not adequately compare current findings with existing literature. For example, established evidence indicates that exercise can increase IL-6 levels; discrepancies or consistencies with prior findings should be critically discussed.
• The discussion section does not fully address potential contradictions or alternative explanations for the observed results.

Author Response

Reviewer　2　

 

This manuscript investigates the effects of diet restriction and diet restriction combined with exercise on fatty pancreas morphology and function in WBN/Kob-Lepr fa/fa rats. The authors conclude that fatty deterioration is not inherently pro-inflammatory and that exercise is required to normalize serum FFA levels and restore subcellular architecture.

However, several conceptual, methodological, and interpretational limitations need to be addressed before the manuscript can be considered for publication.

 

The study lacks an exercise-only group, preventing clear discrimination between the independent effects of exercise and the interaction effects of exercise combined with diet restriction.

 

Thank you for highlighting this. As you correctly noted, it was not feasible to include an exercise‑only group in this study. Obese rats with leptin deficiency exhibit marked skeletal muscle atrophy and characteristically do not engage in voluntary exercise; therefore, it was technically difficult to establish an exercise‑only condition under our experimental design. Therefore, we avoided using expressions such as “the effects of exercise” in the manuscript and instead consistently “the addition of chronic exercise” or “chronic exercise combined with diet restriction” to prevent any misunderstanding. The independent effects of exercise alone, using non‑voluntary exercise modalities, remain an important topic for future investigation. This point has been added to the Limitations section.　 Limitations L404–414

 

Only male rats were included. Given that diabetes and metabolic dysfunction are sex-dependent conditions, inclusion of female animals would strengthen the study and improve generalizability.

 

Thank you for this insightful comment. As you highlighted, only male rats were included in this study. Although WK rats are known to spontaneously develop chronic pancreatitis, its incidence is markedly lower in females, and female WKF rats are not commercially available. Therefore, it was not feasible to include female animals in this study.

We acknowledge that this limitation limits the generalizability of our findings; therefore, in the manuscript, we have clarified that our results represent the effects of interventions in obese male rats that develop pancreatitis.

Future studies using alternative animal models that include females will be necessary to evaluate sex‑dependent differences in pancreatic injury and responses to non‑pharmaceutical interventions. This point has been added to the Limitations section.　 Limitations L404–414

 

 

The 6-week intervention may not adequately capture long-term progression of fatty pancreas and inflammation.

 

Thank you for this important comment. As you noted, sampling was performed at 12 weeks of age after a 6‑week intervention. At this young age, Lean rats had not yet developed pancreatitis or diabetes, whereas Obese rats already exhibited hyperglycemia and early signs of pancreatitis at 12 weeks, allowing us to compare how obesity and ectopic fat accumulation accelerate the onset of metabolic and inflammatory abnormalities. This design enabled us to evaluate whether non‑pharmaceutical interventions could suppress these early pathological changes, and the results were consistent with our hypothesis.

 

Our previous studies demonstrated that various metabolic and structural changes can be detected within a 6‑week intervention period, and this study followed the same established experimental framework. Additionally, in this study effects of adding exercise -such as improvements in skeletal muscle development, mitochondrial function, and reductions in hepatic fat accumulation- were clearly observed, as shown in the supplementary data.

 

However, as you correctly mentioned, this study could not address how these interventions influence the long‑term progression of lipotoxicity, inflammation, or age‑related pancreatic deterioration. For this reason, we did not attempt to draw conclusions regarding the duration of intervention required for sustained benefit. Long‑term studies and investigations in older animals will be necessary to clarify these aspects. We have added this point to the Limitations section of the manuscript.  Limitations L404–414

 

Conclusions regarding implications for human metabolic dysfunction-associated fatty pancreas disease may be overstated, given the specific genetic background of the animal model used.

 

Thank you for highlighting this. In accordance with your comment, we have revised the manuscript to moderate any statements that may overinterpret our findings in relation to improvements in human metabolic dysfunction‑associated fatty pancreas disease. We have also removed Figure 6, which was originally intended to illustrate the potential implications for human pathophysiology.  Discussion (previous version of the manuscript) pvL311–341

 

The manuscript does not clearly describe how the pancreas was harvested and whether a standardized method was used to ensure consistent tissue collection for weight measurements.

 

Thank you for this important comment. We have added a detailed description of the pancreas-harvesting procedure to the Methods section. Material and Methods L449–450  

All pancreatic sampling was performed by the same preclinical pancreatic researcher following a standardized protocol to ensure consistency regarding tissue collection and weight measurements. Specifically, the entire milky‑white pancreatic tissue located below the spleen and adjacent to the small intestine, along with the surrounding tissue, was carefully removed, washed in physiological saline, and separated from low‑density tissue components to isolate the whole pancreas.　

 

The manuscript reports decreased pancreatic protein content in obese rats without providing mechanistic explanation or supporting references.

 

Thank you for this helpful comment. In response to your suggestion, we have substantially expanded our description and discussion of exocrine pancreatic indicators in the manuscript. We added supporting references describing the reduction in the pancreatic protein content and enzyme activities in obesity and diabetes, as well as studies demonstrating the beneficial effects of exercise on exocrine pancreatic function. These additions clarify the mechanistic background and provide appropriate context for the decreased pancreatic protein content observed in obese rats.　Discussion L354–389

 

Inflammation, ER stress, and autophagy are complex processes involving multiple signaling pathways. Assessment of only a single marker for each process is insufficient to conclude that these pathways were comprehensively evaluated.

 

Thank you for this insightful comment. In this study, we evaluated only one or two markers for each pathway related to inflammation, ER stress, and autophagy. As you correctly highlighted, these limited markers are insufficient for comprehensively assessing such complex biological processes involving multiple signaling pathways. Nevertheless, we believe that these molecular findings complement the morphological observations obtained from histology and electron microscopy.

 

We agree that further investigation is needed to elucidate the mechanistic involvement of these pathways in the protective effects of non‑pharmaceutical interventions. We have expanded the Discussion to address the roles of autophagy and ER stress and have added statements acknowledging the limitations of interpreting these pathways based on a minimal set of indicators.　Discussion L294–327

 

The manuscript does not adequately explain the roles of LC3-I or LC3-II in autophagy and interpret the findings accordingly.

 

Thank you for highlighting this. We understand that there are functional differences between LC3‑I and LC3‑II. In our analysis, we also examined the LC3‑ II /LC3‑ I ratio, which followed a pattern that of consistent with the LC3‑II results. For this reason, we presented only the LC3‑II data in our manuscript. As noted in our response to the previous comment, we have expanded the Discussion to provide additional context regarding the interpretation of autophagy in this study.

Discussion L294–327

 

The term "Pancreatic B cells" is potentially misleading, as B cells commonly refer to immune cells. The manuscript should clarify whether pancreatic beta cells are intended and revise terminology accordingly.

 

Thank you for highlighting this. In accordance with your suggestion, we have revised the terminology throughout the manuscript and uniformly replaced “pancreatic B cells” with “pancreatic β‑cells” to avoid confusion with immune B cells.

 

Only serum FFA and triglycerides were measured. No detailed assessment of pancreatic lipid species or lipotoxic intermediates was performed.

 

Thank you for this comment. In this study, we did not measure lipid metabolic indicators within pancreatic tissue. Therefore, we did not discuss the detailed aspects of pancreatic lipid metabolism in our manuscript. We acknowledge that detailed assessment of pancreatic lipid species and lipotoxic intermediates is important for understanding the mechanisms underlying fatty pancreas. These assessments remain an important topic for future investigation.

 

Electron microscopy findings are descriptive without morphometric quantification of mitochondrial damage, ER dilation, or lipid droplet accumulation.

 

Thank you for this valuable comment. In this study, representative electron microscopy images were presented for each group. For electron microscopic observation, pancreatic tissues from 3 animals per group were examined. From each group, more than 10 randomly selected independent longitudinal images of pancreatic acinar cells were analyzed to evaluate the ultrastructure of the mitochondria, the endoplasmic reticulum, and lipid droplets. Morphological quantification analysis of electron microscopic findings will be addressed in future studies.

 

The designation of "early pancreatitis" lacks standardized pathological scoring and fibrosis evaluation.

 

Thank you for this important comment. Standardized diagnostic criteria for chronic pancreatitis have been established in humans, including characteristic imaging findings, histological features, and the following clinical items: (1) repeated upper abdominal or back pain, (2) abnormal serum or urine pancreatic enzyme levels, (3) abnormal pancreatic exocrine function, (4) continued heavy alcohol consumption, and (5) a past history of acute pancreatitis. A diagnosis of early chronic pancreatitis is made when two of items (1) to (5) are positive and imaging findings suggestive of early chronic pancreatitis are present.

 

However, as far as we could determine, no equivalent diagnostic criteria for rats have been established. The WKF rat used in this study is a model that spontaneously develops pancreatitis. In the Obese group, we observed several hallmark features of pancreatitis, including abnormal acinar morphology, inflammatory cell infiltration, marked reductions in exocrine pancreatic indicators, elevated serum pancreatic amylase, and impaired β‑cell function. Based on these findings, we described these animals as having developed pancreatitis.

 

However, as a key feature of advanced chronic pancreatitis–namely, extensive fibrosis–was not observed, we interpreted the condition as representing an early stage of pancreatitis. For this reason, we did not use the term “early pancreatitis” in the manuscript but instead referred to the findings as indicative of an early stage of pancreatic injury.

(Evidence-based clinical practice guidelines for chronic pancreatitis 2021, https://doi.org/10.1007/s00535-022-01911-6)

 

Effects attributed to diet restriction or exercise may partly reflect differences in body weight and metabolic load that were not fully controlled.

 

Thank you for this comment. As you noted, individual variability is inherent in animal experiments, and it is difficult to fully control all experimental conditions, including body weight and metabolic load. In this study, we interpreted the findings based on comparisons between groups while considering this variability. However, because the analyses were conducted with a minimal number of animals, caution is required when extrapolating the results to humans or generalizing the findings more broadly. We added a statement to the Limitations section noting that the analyses were conducted with a limited number of animals.

Limitations L411

 

The units used for glucose and insulin in the HOMA-IR formula appear inconsistent with standard methodology, raising concerns about the validity of insulin resistance assessment.

 

Thank you for your comment. We have corrected the formula and revised the corresponding graph accordingly (HOMA-IR in Figure 1).　Material and Methods L463-464

 

The manuscript does not specify how western blot data were normalized for loading (e.g., housekeeping protein, total protein staining).

 

Thank you for your comment. Although normalization using housekeeping proteins (such as GAPDH or β‑actin) or total protein staining (e.g., CBB staining) is commonly performed to correct for loading variability, the original images (attached) show that staining intensity does not necessarily remain constant even when equal amounts of total protein are applied to each lane. As such variability may introduce additional bias, we chose to present the Western blot data without normalization. All samples were loaded with equal total protein amounts confirmed prior to electrophoresis, and the results were interpreted accordingly.

 

Original, uncropped western blot images are not included, limiting assessment of data integrity.

 

Thank you for this comment. The original, uncropped western blot images have already been provided as requested.

 

The manuscript does not clearly state whether allocation, outcome assessment, or histological analyses were performed in a blinded and randomized manner.

 

Thank you for this comment. In response to your suggestion, we have clarified the procedures related to allocation, outcome assessment, and histological analyses. The group allocation, method, including matching of initial body weight, has been described. The primary outcome has been explicitly stated, and the blinding procedures for histological analyses have been added. Based on these revisions, we have updated the corresponding items in the ARRIVE checklist for resubmission. Material and Methods L422–428, 454–457, 498–500

 

The conclusion that fatty deterioration is not inherently pro-inflammatory is not sufficiently supported by the limited inflammatory assessment and single time point analysis.

 

Thank you for this important comment. As you highlighted, our previous conclusion involved a degree of inference. In fact, compared with the Lean group, the Obese rats exhibited several characteristic features associated with lipid metabolic abnormalities and ectopic fat accumulation, including structural abnormalities in pancreatic tissue and clear inflammatory changes. We have revised the conclusion and moderated the wording to avoid overinterpretation.

Conclusions L519–532

 

Claims regarding skeletal muscle-pancreas crosstalk and myokine involvement are speculative and not directly supported by experimental data.

 

Thank you for this comment. As you mentioned, our statements regarding skeletal muscle–pancreas crosstalk and the involvement of myokines were speculative and not directly supported by experimental data. We therefore consider these aspects to be subjects for future investigation. In response to your suggestion, we have removed the sections of the Discussion that referred to crosstalk and myokine involvement.  

Discussion (previous version of the manuscript) pvL307–341

 

The discussion does not adequately compare current findings with existing literature. For example, established evidence indicates that exercise can increase IL-6 levels; discrepancies or consistencies with prior findings should be critically discussed.

 

Thank you for this valuable comment. We have added relevant references and expanded the Discussion to better integrate our findings with the existing literature, including studies on pancreatitis and IL‑6, exercise‑induced changes in IL‑6, autophagy and ER stress, as well as biochemical and mitochondrial adaptations in skeletal muscle. Regarding IL‑6, increases observed during exercise-particularly resistance or eccentric exercise-are generally attributed to muscle‑damage-induced inflammation. In contrast, moderate physical activity that does not cause substantial muscle damage is less likely to elevate IL‑6 levels. In the revised manuscript, we discuss previous reports showing that aerobic exercise training suppresses pro‑inflammatory markers elevated by a high‑fat diet, and we have interpreted our findings in this context.

Discussion L295–300 (IL-6), Discussion L262-414

 

The discussion section does not fully address potential contradictions or alternative explanations for the observed results.

Thank you for this comment. We have expanded the Discussion to address potential contradictions and alternative explanations for the observed results. For example, although exercise is generally considered to enhance autophagy and alleviate ER stress, we did not observe additional effects when exercise was combined with diet restriction. Similarly, despite the elevation of autophagy-related markers in obese rats, structural deterioration at the tissue and cellular levels was still evident. These points have now been incorporated into the revised Discussion to provide a more balanced and critical interpretation of the findings. Discussion L294-327

 

 

As described above, we revised the manuscript according to your comments. In addition, following the comments from Reviewer 1, we made the following revisions.

We described the characteristics of the db/db obese mouse model in the Introduction and discussed relevant study using this model in the Discussion.　

Introduction L82–86, Discussion L317–320