Lacticaseibacillus paracasei MG5012 and Bifidobacterium animalis subsp. lactis MG741 Alleviate Metabolic Dysfunction-Associated Steatotic Liver Disease and Preserve Skeletal Muscle Integrity in High-Fat-Diet-Fed Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The submitted manuscript presents a well-designed study investigating the effects of two probiotic strains, L. paracasei MG5012 and B. animalis subsp. lactis MG741, on the development of MASLD and the preservation of skeletal muscle integrity in high-fat-diet-fed mice. The Authors apply a wide range of biochemical, histological and molecular analyses, it gives the study a solid experimental foundation. The results showing improvements in liver biomarkers, reductions in hepatic steatosis, modulation of SREBP-1c and FAS expression, and increased GLP-1 levels are important strengths.
However, I have several concerns regarding the discussion section. In its current form, it draws conclusions that appear premature and not fully supported by the presented data:

1. In the first part of the discussion, the Authors state that their study significantly expands the mechanistic scope of previous work, and then cite GLP-1 modulation and lipogenesis regulation as key elements of probiotic action. However, despite emphasizing the role of GLP-1 at many stages of the discussion, the authors do not present any data that mechanistically support this hypothesis. There are many passages such as “it can be concluded” that MG5012 and MG741 improve lipid functions through GLP-1 modulation, which are highly speculative. Even the discussion and previous studies are not conclusive in this regard. The paper lacks an assessment of AMPK pathway activation and GLP-1R expression, and a study using GLP-1 receptor inhibitors would be very easy to prove. The comparison of your own data with the cited literature is, of course, correct, but it does not prove the proposed mechanism of action in the studied model. Please support the discussion with a diagram of the mechanisms. 

2.  In the next section, the authors state that probiotics increase insulin sensitivity in the liver and regulate lipogenesis, as indicated by changes in ALT, AST, and γ-GTP levels. However, these parameters are markers of hepatocyte damage and not direct indicators of insulin resistance. The study did not analyze metabolic indicators, blood glucose levels, the expression of key signaling proteins, etc., so the claim of improved insulin sensitivity is therefore unjustified in its current form.

3. The data refer exclusively to the liver, while the authors repeatedly refer to muscles in their discussion, even though no relevant protein measurements were performed in muscles. Thus, the discussion of the mechanistic effect of probiotics on the antioxidant defense of muscles goes beyond the results presented in this manuscript. Has the suggestion that GLP-1 regulates muscle metabolism been documented experimentally?

4. Another issue concerns the statement that the effects of the strains are systemic and may involve the gut-liver-muscle axis. Although this concept is interesting, biologically justified, and very much in line with current research trends, the authors did not present any data on the gut microbiota, bacterial metabolites, changes in intestinal barrier permeability, bioavailability indicators, etc. Thus, the authors discuss something that is not directly reflected in the results presented in the paper.

Author Response

REVIEW 1

The submitted manuscript presents a well-designed study investigating the effects of two probiotic strains, L. paracasei MG5012 and B. animalis subsp. lactis MG741, on the development of MASLD and the preservation of skeletal muscle integrity in high-fat-diet-fed mice. The Authors apply a wide range of biochemical, histological and molecular analyses, it gives the study a solid experimental foundation. The results showing improvements in liver biomarkers, reductions in hepatic steatosis, modulation of SREBP-1c and FAS expression, and increased GLP-1 levels are important strengths.
However, I have several concerns regarding the discussion section. In its current form, it draws conclusions that appear premature and not fully supported by the presented data:

1. In the first part of the discussion, the Authors state that their study significantly expands the mechanistic scope of previous work, and then cite GLP-1 modulation and lipogenesis regulation as key elements of probiotic action. However, despite emphasizing the role of GLP-1 at many stages of the discussion, the authors do not present any data that mechanistically support this hypothesis. There are many passages such as “it can be concluded” that MG5012 and MG741 improve lipid functions through GLP-1 modulation, which are highly speculative. Even the discussion and previous studies are not conclusive in this regard. The paper lacks an assessment of AMPK pathway activation and GLP-1R expression, and a study using GLP-1 receptor inhibitors would be very easy to prove. The comparison of your own data with the cited literature is, of course, correct, but it does not prove the proposed mechanism of action in the studied model. Please support the discussion with a diagram of the mechanisms. 

A: We sincerely appreciate the reviewer’s insightful and constructive comments regarding the mechanistic link between GLP-1 modulation and lipid metabolism. We agree that providing direct evidence for the signaling pathway is crucial for strengthening our hypothesis.

1) GLP-1/GLP-1R and AMPK-Independent Mechanisms: In response to the reviewer’s suggestion, we performed additional Western blot analysis for p-AMPK/AMPK (Figure F and I). Although the groups treated with MG5012 and MG741 showed an increasing trend in the p-AMPK/AMPK ratio compared to the HFD-only group, the difference did not reach statistical significance (p > 0.05). This suggests that while AMPK activation may partially contribute to lipid metabolism regulation, it might not be the sole mediator in this specific model.

Regarding the GLP-1/GLP-1R mechanism, we acknowledge that direct GLP-1R inhibition would provide more definitive proof. However, given the significant increase in GLP-1 and the concurrent downregulation of SREBP-1 and FAS (Figure G, H), we have revised the Discussion to include AMPK-independent pathways. Specifically, we discuss how GLP-1 can modulate lipogenesis through the cAMP-PKA signaling axis, a well-recognized alternative pathway for SREBP-1c suppression.

2) Schematic Diagram: In response to the reviewer’s request, we have added a schematic diagram (Figure 7) in the conclusion section. This diagram illustrates the proposed mechanism, integrating GLP-1 modulation, SREBP-1/FAS regulation, and the potential signaling crosstalk to provide a clearer overview of the probiotic action.

2. In the next section, the authors state that probiotics increase insulin sensitivity in the liver and regulate lipogenesis, as indicated by changes in ALT, AST, and γ-GTP levels. However, these parameters are markers of hepatocyte damage and not direct indicators of insulin resistance. The study did not analyze metabolic indicators, blood glucose levels, the expression of key signaling proteins, etc., so the claim of improved insulin sensitivity is therefore unjustified in its current form.

A: We sincerely appreciate the reviewer’s precise correction. We fully agree that ALT, AST, and γ-GTP are biomarkers of hepatocellular injury and not direct indicators of insulin resistance.

However, we would like to clarify that our conclusion regarding the improvement of metabolic regulation and insulin sensitivity was primarily based on the Serum Insulin levels (Figure 2G) and GLP-1 levels (Figure 2D), rather than on the liver enzymes. As shown in Figure 2G, the HFD group exhibited significant hyperinsulinemia (a hallmark of insulin resistance), which was markedly attenuated by MG5012 and MG741 treatment (p < 0.05). This reduction in fasting serum insulin, concurrently with the restoration of GLP-1 levels, strongly suggests an improvement in systemic metabolic regulation.

To address the reviewer’s concern and avoid any misunderstanding, we have carefully revised the Discussion section to clearly distinguish between these two effects:

• Metabolic Regulation: We cited the reduction in serum insulin and the restoration of GLP-1 as evidence for improved systemic metabolic regulation and the alleviation of hyperinsulinemia.
• Hepatoprotection: We discussed the reduction in ALT, AST, and γ-GTP solely as indicators of the strains' hepatoprotective effects against HFD-induced liver injury.

3. The data refer exclusively to the liver, while the authors repeatedly refer to muscles in their discussion, even though no relevant protein measurements were performed in muscles. Thus, the discussion of the mechanistic effect of probiotics on the antioxidant defense of muscles goes beyond the results presented in this manuscript. Has the suggestion that GLP-1 regulates muscle metabolism been documented experimentally?

A: We appreciate the reviewer’s insightful comment regarding the mechanistic link between GLP-1 and skeletal muscle. We acknowledge that while we measured GLP-1 levels in serum and liver, we did not measure GLP-1 or its receptor expression directly in skeletal muscle tissue.

1) Systemic Effect of Serum GLP-1: As shown in Figure 2D and 3E, the probiotic treatment significantly restored serum and liver GLP-1 levels. Since GLP-1 is a circulating incretin hormone, its elevation suggests a systemic effect that can reach peripheral tissues, including skeletal muscle.

While we did not measure GLP-1 in skeletal muscle tissue, we did perform Western blot analysis on skeletal muscle tissue to measure downstream effector proteins—specifically, antioxidant enzymes (SOD, CAT, GPx; Figure 6F-I). The significant upregulation of these proteins in the muscle tissue provides experimental evidence of improved muscle health.

2) Literature Support: The regulation of muscle metabolism by GLP-1 has been documented experimentally in previous studies [30], which showed that GLP-1 receptor signaling enhances mitochondrial function and glucose uptake in muscle.

Therefore, in the revised Discussion, we have carefully rephrased our conclusion to avoid overstatement. We now suggest that the systemic elevation of serum GLP-1, combined with the observed upregulation of muscle antioxidant enzymes, supports the hypothesis of a beneficial gut-liver-muscle axis, consistent with established literature.

4. 
   Another issue concerns the statement that the effects of the strains are systemic and may involve the gut-liver-muscle axis. Although this concept is interesting, biologically justified, and very much in line with current research trends, the authors did not present any data on the gut microbiota, bacterial metabolites, changes in intestinal barrier permeability, bioavailability indicators, etc. Thus, the authors discuss something that is not directly reflected in the results presented in the paper.

A: We agree that direct evidence supporting the "gut" component of the axis was insufficient in the original manuscript. To address this concern and provide experimental evidence for the improvement of intestinal barrier integrity, we have included new Western blot data measuring the expression of tight junction proteins, Occludin and Claudin-1, in colonic tissue.

Intestinal Barrier Integrity (Figure 7): As shown in the newly added Figure X, HFD feeding significantly reduced the protein expression of Occludin and Claudin-1 compared to the ND group, indicating impaired intestinal barrier function. However, supplementation with MG5012 and MG741 significantly restored the expression of these tight junction proteins.

Therefore, we have added these results to the revised manuscript to substantiate our discussion on the gut-liver-muscle axis. While we did not perform microbiome sequencing in this study, the clear improvement in gut barrier integrity (Occludin/Claudin-1) combined with the systemic metabolic benefits provides a solid biological justification for the proposed axis.

Reviewer 2 Report

Comments and Suggestions for Authors Methods:
1. Animals: Were the mice singly housed? How was the probiotic administered to the mice orally (gavage, self-administration, or drinking water)?
2. Serum Biochemistry: How were CPK and LDH assessed?
3. Histological Analysis: This paragraph is confusing, with the steps for the muscle analysis written in between the liver analysis. Also, since this is a MASLD paper, it would be more relevant if other histological features of MASLD are evaluated, such as fibrosis, ballooning, and inflammation. Can the authors also write how many high-power objective fields per mouse were analyzed?
4. ELISA: Can the authors provide the catalog numbers and assay performance parameters for the different analyses? Is the GLP-1 assay detecting the active GLP-1 peptide?
5. Western Blot: Can the authors provide the catalog numbers and the dilutions used for the primary antibodies?
6. Statistics: Which indicate that the HFD group served as the control for the Dunnett.
Results:
1. Can the authors provide the absolute food intake?
2. Is there data for the measurement of adiposity?
3. Only some of the original blots (n=3/group) for the MnSOD, Catalase, Gpx, and Actin were provided.
4. Is the liver weight expressed per unit body weight of the mouse?
5. Why was sylimarin not included in the adipocyte histology and muscle analysis?
6. Was lean mass assessed by weighing one muscle group? If so, please indicate this so as not to overgeneralize that there was a systemic sarcopenia. Also, the animal model does not reflect sarcopenia.
7. Do these probiotics change energy expenditure?
Discussion
1. The HFD-fed mouse model is not the chosen model of MASLD. Typically, it is the Western Diet with 2% cholesterol, which is the standard MASLD diet. Why was this model used?
2. LDH is a bi-directional enzyme that converts lactate to pyruvate and vice versa, and is ubiquitously expressed. Thus, its serum activity cannot be used as a proxy for muscle health.
3. No sarcopenia was observed in the HFD mice, which is also not associated with the changes in the biochemical analysis of muscle. Therefore, the improvements in the CSA might not be due to obesity-induced sarcopenia. What could be the reason why these mice have larger muscle fibers? Was there a difference in the voluntary activity of the probiotic groups?
4. A large portion of the discussion was about the role of GLP-1, yet no discussion about food intake and body weight was included. Improvements in metabolism due to GLP-1 are mostly driven by reduced appetite and body weight in humans. Can the authors comment on the potential implications of the lack of effect of these probiotics on these two parameters, yet are still able to exert benefits to metabolism?
5. Can the authors comment on a potential mechanism by which these two bacteria increase GLP-1, either increased secretion or reduced degradation?
6. How do these probiotics compare with other probiotics in terms of improving hepatic and muscle health?
Overall
1. The authors use strong words that are usually used in studies proving causation. I advise the authors to use more gentle words that apply to association studies.
2. Many of the citations are reviews. I would suggest that the authors cite the original research article.

Author Response

REVIEW 2

Methods:
1. Animals: Were the mice singly housed? How was the probiotic administered to the mice orally (gavage, self-administration, or drinking water)?

A: We appreciate the reviewer’s query regarding the details of animal housing and administration methods.

1) Housing: To clarify, the mice were housed in groups of 3–5 per cage (not singly housed) under controlled conditions.

2) Administration: The probiotics and silymarin were suspended in sterile PBS and administered via oral gavage (using a feeding needle) to ensure precise dosage delivery, rather than through drinking water or self-administration.

2. Serum Biochemistry: How were CPK and LDH assessed?

A: We assessed the serum levels of CPK and LDH using the same automated biochemical analyzer (Hitachi 3500, HITACHI, Tokyo, Japan) used for other metabolic markers (AST, ALT, etc.). To avoid confusion regarding the methodology, we have revised Section 2.3 (Serum Biochemical Analysis) to explicitly list CPK and LDH along with the other parameters measured by the Hitachi 3500 system.

3. Histological Analysis: This paragraph is confusing, with the steps for the muscle analysis written in between the liver analysis. Also, since this is a MASLD paper, it would be more relevant if other histological features of MASLD are evaluated, such as fibrosis, ballooning, and inflammation. Can the authors also write how many high-power objective fields per mouse were analyzed?

A: We have reorganized Section 2.4 by separating it into two distinct paragraphs: "Liver Histological Analysis" (including both H&E and Oil Red O staining) and "Skeletal Muscle Histological Analysis." This restructuring ensures a logical flow and clarity.

Also, we have added the specific number of analyzed fields to the revised manuscript. As stated in the updated text, we analyzed at least three to five randomly selected fields per mouse for both the quantification of ORO-positive areas (liver) and muscle fiber CSA (muscle) using ImageJ software.

4. ELISA: Can the authors provide the catalog numbers and assay performance parameters for the different analyses? Is the GLP-1 assay detecting the active GLP-1 peptide?

A: In response to your suggestion, we have compiled the manufacturers, catalog numbers, and assay performance parameters (e.g., detection range and sensitivity) for all ELISA kits used in this study. This information is now provided in Supplementary Table S1.

5. Western Blot: Can the authors provide the catalog numbers and the dilutions used for the primary antibodies?

A: In response to your suggestion, we have compiled the full list of primary antibodies, including their manufacturers, catalog numbers, and specific dilution ratios used in this study. This information is now provided in Supplementary Table S2. We have also updated Section 2.6 (Western Blot Analysis) in the revised manuscript to reference this table.

6. Statistics: Which indicate that the HFD group served as the control for the Dunnett.

A: As suggested, we have revised the "2.7. Statistical Analysis" section to explicitly state that the HFD group served as the control for Dunnett’s multiple comparisons test to evaluate the efficacy of the treatments.

Results:
1. Can the authors provide the absolute food intake?

A: We monitored the daily food intake throughout the experimental period to ensure that the anti-obesity effects of the probiotics were not attributed to appetite suppression or issues with palatability.

As shown in the figure above (provided here for the reviewer's reference), the absolute food intake was significantly lower in the HFD-fed groups compared to the ND group, which is a typical compensatory mechanism due to the high caloric density of the HFD. However, importantly, there were no significant differences in food intake among the HFD, Silymarin, and probiotic-treated groups (MG5012 and MG741).

Since the caloric intake was consistent across the HFD groups, the reduction in body weight gain in the probiotic groups suggests an improvement in metabolic regulation rather than reduced energy intake. Therefore, we believe that the Food Efficiency Ratio (FER), which normalizes weight gain to food intake (Figure 1D), serves as a more representative and mechanistically relevant index for this study. Accordingly, we have maintained the FER data in the manuscript while providing this raw data for your confirmation.

2. Is there data for the measurement of adiposity?

A: To comprehensively assess adiposity, we measured both the mass of the epididymal fat pads and performed a histomorphometric analysis of adipocyte size.

We have added the data for epididymal fat weight in Supplementary Figure S1. As shown in the figure, while there was a slight decreasing trend in the probiotic-treated groups compared to the HFD group, the difference in absolute fat mass did not reach statistical significance.

However, since adipose tissue dysfunction in obesity is characterized more critically by adipocyte hypertrophy than by mass alone, we focused on the histological analysis. Treatment with MG5012 and MG741 significantly reduced the adipocyte cross-sectional area (CSA) (p < 0.01 and p < 0.05, respectively) compared to the HFD group.

These results suggest that although the probiotics did not drastically reduce the total fat mass within the experimental period, they significantly ameliorated adipocyte hypertrophy, indicating an improvement in adipose tissue quality and metabolic health. We have updated the Results section to include the fat weight data and clarify this interpretation.

3. Only some of the original blots (n=3/group) for the MnSOD, Catalase, Gpx, and Actin were provided.

A: We apologize for any confusion regarding the availability of the raw data. We have provided the full, uncropped original images for all Western blots (including all replicates for MnSOD, Catalase, GPx, and Actin) as a separate file in the online submission system. Please note that the bands presented in Figure 6 are representative images (n=3) selected for clarity and layout purposes. We kindly ask the reviewer to check the attached raw data file for verification.

4. Is the liver weight expressed per unit body weight of the mouse?

A: The data presented in Figure 4C represent the absolute wet weight of the liver (measured in mg), not normalized to body weight. We chose to present the absolute liver weight for the following reasons:

In the HFD-induced obesity model, body weight increases disproportionately due to excessive fat accumulation. Normalizing organ weight to body weight in such obese animals can sometimes mask the actual extent of organ hypertrophy (hepatomegaly) because the denominator (body weight) is significantly elevated. Therefore, we considered the absolute liver weight to be a more direct and accurate indicator of hepatic steatosis and hepatomegaly in this specific experimental setting.

We have modified the Figure Legend to explicitly state "Absolute liver weight" to avoid any potential confusion for the readers.

5. Why was sylimarin not included in the adipocyte histology and muscle analysis?

A: We appreciate the reviewer’s insightful query. We included Silymarin primarily as a positive control to validate improvements in the Gut-Liver Axis, given its well-established efficacy in protecting against liver injury and restoring intestinal barrier integrity.

• Gut-Liver Axis (Included): Accordingly, we analyzed silymarin in both liver tissues (histology, serum markers) and colonic tissues (tight junction proteins) to benchmark the probiotics' efficacy in ameliorating the core pathology of MASLD.
• Peripheral Tissues (Excluded): However, the analyses of adipocyte histology and skeletal muscle were designed as specific exploratory endpoints to investigate the broader, systemic metabolic potential of the probiotic strains (MG5012 and MG741). Since silymarin is not typically utilized as a standard therapeutic for sarcopenia or adipocyte remodeling in this context, we prioritized the analysis of the probiotic groups against the HFD control to highlight their distinct systemic benefits beyond the gut-liver axis.

6. Was lean mass assessed by weighing one muscle group? If so, please indicate this so as not to overgeneralize that there was a systemic sarcopenia. Also, the animal model does not reflect sarcopenia.

A: We appreciate the reviewer’s accurate observation.

We confirm that the data in Figure 6B represents the weight of the gastrocnemius femoris muscle only, not systemic lean mass. To prevent overgeneralization, we have revised the figure caption and the main text to explicitly state "gastrocnemius muscle weight" instead of general "muscle weight."

We agree that the HFD model does not reflect classical sarcopenia (loss of muscle mass). Accordingly, we do not use the term "sarcopenia" from the manuscript.

Instead, we have reframed the interpretation in Section 3.6 to focus on "muscle integrity" and "quality." We highlight that while the gross muscle weight did not change (Figure 6B), the probiotics significantly improved muscle fiber cross-sectional area (CSA) and antioxidant defense, indicating an amelioration of HFD-induced muscle atrophy and metabolic stress at the cellular level.

7. Do these probiotics change energy expenditure?

A: As detailed in our response to Question 1, the probiotic-treated groups exhibited significantly reduced body weight gain and adiposity despite having similar food intake to the HFD control group. Consequently, the Food Efficiency Ratio (FER) was significantly lower in the MG5012 and MG741 groups (Figure 1D). These findings strongly suggest that the anti-obesity effects of the probiotics are mediated by increased energy expenditure rather than appetite suppression.

Discussion
1. The HFD-fed mouse model is not the chosen model of MASLD. Typically, it is the Western Diet with 2% cholesterol, which is the standard MASLD diet. Why was this model used?

A: While we agree that the Western Diet (high fat, high sugar, and high cholesterol) is an excellent model for inducing advanced NASH, inflammation, and fibrosis, the primary objective of this study was to investigate the efficacy of the probiotic strains in ameliorating metabolic dysfunction (obesity and insulin resistance) and early-stage hepatic steatosis.

Suitability of HFD: The 60% HFD model is a well-established and widely accepted standard for inducing diet-induced obesity, insulin resistance, and hepatic lipid accumulation, which are the fundamental hallmarks of Metabolic dysfunction-associated Steatotic Liver Disease (MASLD).

Since our study aimed to elucidate the systemic metabolic benefits of the probiotics—including their effects on the gut-liver axis, adipose tissue hypertrophy, and skeletal muscle integrity—we selected the HFD model because it robustly mimics the pathophysiology of metabolic syndrome-driven fatty liver, rather than toxicity-induced or advanced fibrotic models.

2. LDH is a bi-directional enzyme that converts lactate to pyruvate and vice versa, and is ubiquitously expressed. Thus, its serum activity cannot be used as a proxy for muscle health.

A: We fully agree with the reviewer’s comment.

We acknowledge that LDH is a ubiquitous enzyme and its serum activity reflects general cellular damage rather than being a specific proxy for skeletal muscle health. However, in this study, LDH was analyzed as a supportive marker alongside CPK, which is a more specific indicator of muscle damage. As shown in Figure 6D, serum CPK levels were significantly reduced in the probiotic groups, providing direct biochemical evidence of reduced muscle injury. Furthermore, histological analysis (Figure 6A & C) confirmed that the probiotics attenuated myofiber atrophy.

Therefore, we interpreted the reduction in LDH not as a standalone measure of muscle health, but as evidence of reduced overall metabolic stress and cellular damage in the context of HFD-induced obesity. This pattern is consistent with the improvements observed in both liver (AST/ALT) and muscle (CPK) tissues.

We have revised the relevant text in Section 3.6 to reflect this more accurate interpretation.

3. No sarcopenia was observed in the HFD mice, which is also not associated with the changes in the biochemical analysis of muscle. Therefore, the improvements in the CSA might not be due to obesity-induced sarcopenia. What could be the reason why these mice have larger muscle fibers? Was there a difference in the voluntary activity of the probiotic groups?

A: We acknowledge that we did not quantitatively measure voluntary physical activity in this study. Therefore, we cannot rule out the possibility that the probiotics influenced behavioral activity. However, given the significant biochemical changes observed, we believe the primary mechanism is metabolic rather than behavioral.

We agree that HFD mice did not exhibit classical sarcopenia (muscle mass loss). However, HFD is known to induce "obesity-related muscle atrophy" or myosteatosis at the cellular level, primarily driven by lipotoxicity and oxidative stress, which leads to protein degradation and shrinkage of muscle fibers.

As shown in Figure 6F–I, the probiotic treatments (especially MG741) significantly upregulated the expression of antioxidant enzymes (SOD, CAT, GPx) in skeletal muscle. Therefore, we interpret the larger CSA in the probiotic groups not as "hypertrophy" induced by exercise, but as the preservation of muscle integrity against HFD-induced oxidative damage and atrophy. The probiotics effectively blocked the catabolic signals triggered by the high-fat diet, thereby maintaining healthy fiber size.

4. A large portion of the discussion was about the role of GLP-1, yet no discussion about food intake and body weight was included. Improvements in metabolism due to GLP-1 are mostly driven by reduced appetite and body weight in humans. Can the authors comment on the potential implications of the lack of effect of these probiotics on these two parameters, yet are still able to exert benefits to metabolism?

A: We thank the reviewer for this crucial observation. We agree that GLP-1 is classically known to improve metabolism largely through central appetite suppression and subsequent weight loss. However, emerging evidence suggests that GLP-1 also exerts direct peripheral metabolic effects independent of its anorectic actions.

In our study, while absolute food intake did not decrease, the probiotic-treated groups showed significantly reduced body weight gain and adiposity, leading to a decreased Food Efficiency Ratio (FER). This indicates an increase in energy expenditure.

We interpret this as the probiotics-induced GLP-1 enhancing metabolic flexibility in peripheral tissues. Specifically, GLP-1 signaling is known to promote glucose uptake and mitochondrial biogenesis in skeletal muscle and lipid oxidation in the liver.

Therefore, the metabolic benefits observed in our study (improved insulin sensitivity, reduced steatosis, preserved muscle integrity) despite unchanged food intake are likely driven by these direct peripheral actions of GLP-1, rather than central appetite regulation.

We have expanded the Discussion section to address this distinction, citing relevant literature.

5. Can the authors comment on a potential mechanism by which these two bacteria increase GLP-1, either increased secretion or reduced degradation?

A: We have revised the Conclusion to address the potential mechanism of GLP-1 elevation. While we did not directly measure DPP-4 activity, previous studies suggest that probiotics and their metabolites (e.g., SCFAs) primarily stimulate enteroendocrine L-cells to secrete GLP-1. Therefore, we have updated the text to state that the observed effects are likely driven by "promoting the secretion of GLP-1 from intestinal L-cells.

6. How do these probiotics compare with other probiotics in terms of improving hepatic and muscle health?

A: We acknowledge that probiotic effects are highly strain-specific. While many probiotics have been documented to improve hepatic health (Gut-Liver axis), fewer strains have been shown to effectively modulate skeletal muscle health (Gut-Muscle axis).

The distinguishing feature of MG5012 and MG741 compared to other conventional probiotics is their dual efficacy. Our data demonstrates that these strains not only alleviated hepatic steatosis and liver injury but also significantly preserved skeletal muscle integrity (CSA) and enhanced antioxidant defense (SOD, CAT, GPx).

This suggests that MG5012 and MG741 may be more effective than single-target probiotics in managing obesity-related complications, as they address the metabolic dysfunction of both the liver and muscle simultaneously. We have added a paragraph in the Discussion section to elaborate on this strain-specific advantage and comparison with existing literature.

Overall
1. The authors use strong words that are usually used in studies proving causation. I advise the authors to use more gentle words that apply to association studies.

A: We agree with the reviewer’s suggestion. We have carefully reviewed the manuscript and tempered the language to avoid overstating causal relationships. Strong terms have been replaced with more tentative phrasing to more accurately reflect the nature of our findings.

2. Many of the citations are reviews. I would suggest that the authors cite the original research article.

A: In response to your suggestion, we have prioritized citing original experimental research articles rather than reviews for the newly added mechanistic discussions (e.g., regarding GLP-1 mediated muscle protection and oxidative stress) to ensure that our claims are solidly supported by primary data.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors partially addressed the comments after the first round of review, but the revisions are of uneven quality. On the plus side, new results on intestinal barrier integrity and p-AMPK/AMPK analysis have been added, which strengthens the experimental part and better justifies the gut-liver-muscle axis theme. However, a key element of the mechanistic discussion remains insufficiently proven. Conclusions regarding the improvement of insulin sensitivity need to be worded more cautiously, as the study does not present glycemic measurements or tests that would allow for an unambiguous assessment of insulin resistance. Consequently, I recommend clearly separating hypotheses from conclusions directly derived from the data and supplementing the section on study limitations to avoid increasing the credibility of the message.