Review Reports - Peripheral GABA Signaling in Metabolic Adaptation and Maladaptation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled "Peripheral GABA Signaling in Metabolic Adaptation and Maladaptation" discusses the emerging role of peripheral GABA signaling in metabolic regulation and metabolic disease. The topic is timely and potentially valuable, and the manuscript covers biochemical sources, tissue-specific actions, disease-related remodeling, and translational strategies. However, the current version still requires substantial revision in literature selection, concept definition, evidence classification, mechanistic interpretation, figure presentation, and formatting. Specific comments are as follows:

Lines 72-79:The manuscript does not describe how the literature was searched or selected. The authors should provide a brief statement of databases, search period, search terms, and inclusion/exclusion criteria; otherwise, the balance and reproducibility of this review cannot be judged.
Lines 20-38:The abstract uses terms such as adaptive, compensatory, and maladaptive GABA signaling, but these concepts are not clearly defined. Please briefly explain these categories and state the main conclusion of the review more explicitly.
Lines 80-92 and 106-127:The manuscript sometimes treats GABA as a metabolite, extracellular ligand, and exposure marker within the same discussion. These roles should be separated more clearly to avoid conceptual ambiguity.
Lines 128-149:The receptor section remains too general. The authors should connect GABAA/GABAB receptor signaling, chloride gradients, and GPCR-mediated effects to specific peripheral tissues rather than only summarizing receptor biology.
Lines 150-192:The sources of peripheral GABA need clearer classification. Host-derived, dietary, and microbiota-derived GABA should be discussed separately, and the authors should indicate which source-function relationships are experimentally demonstrated and which remain inferred.
Lines 95-104 (Figure 1):Figure 1 is useful but too linear. It should more clearly distinguish GABA sources, metabolic fate, extracellular access, receptor engagement, and downstream functional outputs.
Lines 193-204:A summary table is needed to improve readability. It is recommended to list representative studies by tissue, model, GABA source/intervention, main endpoint, and major limitation.
Lines 217-246:The pancreatic islet section combines evidence from rodent and human studies. The authors should separate these data and clarify whether the evidence supports beta-cell proliferation, insulin secretion, glucagon restraint, or all three.
Lines 247-261 and 377-410:The discussion of hepatic GABA is not sufficiently balanced. The authors need to reconcile the harmful hepatic GABA-output model in obesity with reports suggesting protective or beneficial effects of oral GABA.
Lines 263-281 and 411-460:The adipose tissue section does not clearly identify the direct responding cell types. The authors should specify whether the proposed effects involve adipocytes, immune cells, stromal cells, microbiota-mediated signals, or indirect systemic changes.
Lines 283-300:The immune-cell discussion needs stronger linkage to metabolic outcomes. Please clarify whether the reported immune effects are causally involved in metabolic improvement or are only associated observations.
Lines 301-325:The skeletal muscle section is relatively weak compared with other tissue sections. Unless direct evidence of GABA-responsive signaling in skeletal muscle is provided, this section should be framed as a downstream insulin-sensitive readout.
Lines 326-339:The classification of GABA signaling as adaptive, compensatory, or maladaptive is important but underdeveloped. The authors should provide operational criteria for assigning each tissue-level response to one category.
Lines 340-352 (Figure 3):Figure 3 should distinguish experimentally demonstrated pathways from inferred or hypothetical links. Different arrow styles or labels would make the evidence level clearer.
Lines 461-505:The translational section mixes evidence from obesity, T2DM, prediabetes, and type 1 diabetes. These disease contexts are not equivalent and should be discussed separately to avoid overgeneralization.
Lines 506-536:For hepatic GABA-targeted strategies, the authors should specify measurable biomarkers of pathway engagement, such as hepatic GABA-T activity, hepatic GABA abundance, vagal readouts, fasting insulin, or insulin sensitivity.
Lines 537-580:The adipose-immune-microbiota discussion needs better consideration of confounding factors. Changes in food intake, body weight, microbiota composition, and inflammatory tone may all influence the reported outcomes.
Lines 581-624 and Box 1:The limitations of the current evidence should be stated more directly. In particular, the review should acknowledge the heavy reliance on animal/cell-based evidence, limited human validation, and uncertainty about active GABA sources.
References section (Lines 651-850):The reference format is inconsistent. Please standardize author names, journal titles, capitalization, punctuation, DOI style, and spacing according to the journal guidelines.
Page 1 around Line 43 and the back matter:Several template elements appear incomplete, including editorial placeholders and publication statements. The authors should remove placeholders and complete the funding, conflict-of-interest, data availability, and related declarations before submission.
Lines 20-38, 72-79, 326-339 and 625-645:Most importantly, the review lacks a clear conceptual backbone. The authors repeatedly mention adaptive, compensatory, and maladaptive GABA signaling, but do not develop these terms into their own theoretical model. As a result, the manuscript reads more like a collection of tissue-specific observations than a thesis-driven review. The authors should define a core framework, for example linking GABA source, tissue access, target cell/receptor, disease state, and functional output, and then use this framework consistently throughout the manuscript.

Author Response

Response to Reviewer 2

Comment 1.

Lines 72–79: The manuscript does not describe how the literature was searched or selected. The authors should provide a brief statement of databases, search period, search terms, and inclusion/exclusion criteria; otherwise, the balance and reproducibility of this review cannot be judged.

Response: We thank the reviewer for this thoughtful comment. We respectfully clarify that this manuscript is a narrative, mechanism-focused Review rather than a systematic review or meta-analysis. Accordingly, a formal PRISMA-style search strategy, screening workflow, and quantitative inclusion/exclusion process are not applicable. To improve transparency, we added a paragraph at the end of the Introduction explaining the conceptual framework used to identify and synthesize the literature. The revised text states that the Review evaluates peripheral GABA biology according to GABA source, tissue access, responding cell or receptor pathway, disease state, and metabolic outcome. This addition clarifies the basis for literature selection while preserving the narrative format of the Review.

Comment 2.

Lines 20–38: The abstract uses terms such as adaptive, compensatory, and maladaptive GABA signaling, but these concepts are not clearly defined. Please briefly explain these categories and state the main conclusion of the review more explicitly.

Response: We thank the reviewer for this helpful suggestion. We revised the Abstract to define adaptive, compensatory, and maladaptive GABA signaling more clearly. Peripheral GABA signaling is now presented as a functional continuum in which adaptive responses preserve normal tissue regulation, compensatory responses are recruited during metabolic stress to limit dysfunction, and maladaptive responses reinforce disease-associated physiology. We also strengthened the concluding statement to emphasize the principal conclusion of the Review: peripheral GABA signaling is neither inherently beneficial nor harmful; rather, its metabolic consequences depend on GABA source, tissue context, and disease stage.

Comment 3.

Lines 80–92 and 106–127: The manuscript sometimes treats GABA as a metabolite, extracellular ligand, and exposure marker within the same discussion. These roles should be separated more clearly to avoid conceptual ambiguity.

Response: We appreciate this insightful comment. Section 2 has been revised to distinguish intracellular GABA metabolism, extracellular receptor-mediated signaling, and measurements of GABA exposure. The revised text explains that intracellular GABA functions as a metabolic intermediate within the GABA shunt, extracellular GABA serves as a ligand for GABA receptors on responsive cells, and GABA measured in blood, tissues, intestinal contents, or fecal samples reflects exposure or abundance rather than receptor engagement. Section 2.1 further clarifies that intracellular GABA abundance may reflect synthesis, uptake, storage, or metabolism, whereas receptor-mediated signaling requires extracellular access to an appropriate target cell.

Comment 4.

Lines 128–149: The receptor section remains too general. The authors should connect GABA_A/GABA_B receptor signaling, chloride gradients, and GPCR-mediated effects to specific peripheral tissues rather than only summarizing receptor biology.

Response: We thank the reviewer for this important suggestion. Section 2.2 has been revised to relate receptor biology directly to peripheral physiology. The revised text explains how cell-specific chloride gradients produce distinct GABA_A receptor responses in pancreatic α- and β-cells and discusses tissue-specific metabolic effects of GABA_B receptor signaling, including effects in brown adipose tissue. To avoid unnecessary repetition, detailed organ-specific mechanisms are discussed in the relevant tissue sections. The revised section emphasizes that receptor expression alone is insufficient to define biological function and that receptor responses depend on receptor composition, chloride homeostasis, cell identity, and intracellular signaling context.

Comment 5.

Lines 150–192: The sources of peripheral GABA need clearer classification. Host-derived, dietary, and microbiota-derived GABA should be discussed separately, and the authors should indicate which source–function relationships are experimentally demonstrated and which remain inferred.

Response: We appreciate this recommendation. Section 2.3 has been reorganized into separate subsections covering host-derived, dietary, and microbiota-derived GABA. For host-derived GABA, the revised text summarizes direct evidence of peripheral synthesis, cellular handling, and release while clarifying that detection of GABA or its synthetic machinery establishes source potential rather than biological function. For dietary GABA, we distinguish experimentally demonstrated intestinal absorption, peripheral exposure, and metabolic incorporation from evidence of direct receptor engagement in a defined tissue. For microbiota-derived GABA, we separate demonstrated bacterial GABA production and biological activity in selected experimental models from unresolved questions concerning host access, distal tissue action, and relevance to obesity and type 2 diabetes.

Comment 6.

Lines 95–104 (Figure 1): Figure 1 is useful but too linear. It should more clearly distinguish GABA sources, metabolic fate, extracellular access, receptor engagement, and downstream functional outputs.

Response: We thank the reviewer for this helpful suggestion. Figure 1 has been redesigned to separate GABA sources, intracellular metabolic fate, extracellular access, receptor engagement, and downstream functional outcomes. The revised schematic distinguishes endogenous tissue production, dietary intake, and microbial production; separates intracellular GABA metabolism through GABA transaminase and the GABA shunt from extracellular receptor signaling; and highlights that systemic GABA exposure does not necessarily indicate local receptor engagement. The figure also indicates that downstream effects depend on receptor composition, tissue context, and disease state. The figure legend has been revised accordingly.

Comment 7.

Lines 193–204: A summary table is needed to improve readability. It is recommended to list representative studies by tissue, model, GABA source/intervention, main endpoint, and major limitation.

Response: We thank the reviewer for this helpful suggestion. We added Table 1, which summarizes representative studies according to tissue or compartment, experimental model, GABA source or intervention, principal tissue-linked outcome, and key limitation. The table focuses on the organ-specific actions of peripheral GABA discussed in Section 3, while disease-associated remodeling and therapeutic evidence remain addressed in the later sections. This addition complements the figures and improves comparison of the evidence across tissues.

Comment 8.

Lines 217–246: The pancreatic islet section combines evidence from rodent and human studies. The authors should separate these data and clarify whether the evidence supports β-cell proliferation, insulin secretion, glucagon restraint, or all three.

Response: We appreciate this valuable suggestion. The pancreatic islet section has been revised to distinguish rodent and human evidence more clearly. The revised discussion identifies glucagon restraint as being supported primarily by rodent islet models, distinguishes human evidence for GABA-mediated modulation of β-cell secretory activity, and discusses β-cell proliferation separately as a context-dependent finding from experimental systems. The concluding synthesis now differentiates these three proposed functions and indicates the relative strength of the supporting evidence.

Comment 9.

Lines 247–261 and 377–410: The discussion of hepatic GABA is not sufficiently balanced. The authors need to reconcile the harmful hepatic GABA-output model in obesity with reports suggesting protective or beneficial effects of oral GABA.

Response: We thank the reviewer for this insightful comment. The manuscript now distinguishes the obesity-associated hepatic GABA-output pathway from studies using oral GABA administration. These findings represent distinct sources and routes of peripheral GABA exposure rather than contradictory biological mechanisms. The revised text clarifies that disease-associated hepatic GABA output reflects increased hepatocyte GABA production and release, which can alter hepatic vagal signaling and systemic insulin physiology, whereas oral GABA increases gastrointestinal and circulating exposure that may engage different peripheral tissues or pathways. This distinction reinforces the central conclusion that the effects of peripheral GABA depend on its source, route of availability, site of action, and disease context.

Comment 10.

Lines 263–281 and 411–460: The adipose tissue section does not clearly identify the direct responding cell types. The authors should specify whether the proposed effects involve adipocytes, immune cells, stromal cells, microbiota-mediated signals, or indirect systemic changes.

Response: We thank the reviewer for this insightful comment. We revised the physiological and disease-remodeling sections to clarify the cell populations currently implicated in adipose GABA signaling. The revised text distinguishes direct evidence involving adipocytes, macrophages, stromal cells, and other immune populations from mechanisms likely mediated indirectly through microbiota-derived signals or systemic metabolic changes. We also emphasize that, although several adipose-resident cell populations express components of the GABA signaling machinery, the identity of the primary GABA-responsive cell population remains incompletely resolved. This distinction is now highlighted in the adipose physiology, disease-remodeling, and therapeutic sections.

Comment 11.

Lines 283–300: The immune-cell discussion needs stronger linkage to metabolic outcomes. Please clarify whether the reported immune effects are causally involved in metabolic improvement or are only associated observations.

Response: We thank the reviewer for this important observation. The immune-cell section now distinguishes demonstrated immune-cell responses from causal metabolic outcomes. The revised text clarifies that GABA-responsive changes in cytokine production, migration, and activation state have been directly demonstrated in several immune-cell populations. However, although these changes accompany improved glucose homeostasis and reduced adipose inflammation in some experimental studies, definitive evidence that a specific GABA-responsive immune population mediates the metabolic improvement remains limited. We therefore identify cell-specific manipulation of GABA receptors, transporters, or metabolic pathways as necessary to establish causality.

Comment 12.

Lines 301–325: The skeletal muscle section is relatively weak compared with other tissue sections. Unless direct evidence of GABA-responsive signaling in skeletal muscle is provided, this section should be framed as a downstream insulin-sensitive readout.

Response: We appreciate this important comment. The skeletal muscle subsection has been revised to present skeletal muscle primarily as an insulin-sensitive downstream tissue rather than a site with a well-established local GABA signaling pathway. The revised text emphasizes that hepatic GABA signaling can influence skeletal-muscle glucose uptake through systemic insulin-related mechanisms, while evidence from oral GABA studies supports metabolic incorporation into skeletal-muscle homocarnosine and possible repair-associated effects. We also state explicitly that direct regulation of muscle glucose uptake, insulin signaling, or mitochondrial metabolism by local GABA signaling remains unestablished.

Comment 13.

Lines 326–339: The classification of GABA signaling as adaptive, compensatory, or maladaptive is important but underdeveloped. The authors should provide operational criteria for assigning each tissue-level response to one category.

Response: We thank the reviewer for this insightful suggestion. The opening of Section 4 has been revised to define these categories operationally within the context of metabolic remodeling. Adaptive signaling is described as preserving normal tissue regulation, compensatory signaling as limiting metabolic stress without fully restoring homeostasis, and maladaptive signaling as reinforcing disease-associated dysfunction. These criteria now provide a consistent framework for interpreting tissue-specific GABA responses as obesity and type 2 diabetes progress.

Comment 14.

Lines 340–352 (Figure 3): Figure 3 should distinguish experimentally demonstrated pathways from inferred or hypothetical links. Different arrow styles or labels would make the evidence level clearer.

Response: We thank the reviewer for this helpful recommendation. Figure 3 already distinguishes the nature of the depicted relationships through its graphical conventions. As specified in the figure legend, solid arrows indicate downstream physiological outputs, dashed arrows indicate proposed or indirect GABA-related signaling, and double-headed arrows indicate reciprocal communication. We therefore retained the current figure design because it already separates established downstream relationships from inferred or indirect pathways while preserving visual clarity. The accompanying text also uses cautious terminology, including “associated with,” “linked to,” and “proposed,” where causal evidence remains incomplete.

Comment 15.

Lines 461–505: The translational section mixes evidence from obesity, type 2 diabetes, prediabetes, and type 1 diabetes. These disease contexts are not equivalent and should be discussed separately to avoid overgeneralization.

Response: We thank the reviewer for this important observation. We retained the pathway-based organization of the Review but revised the text to distinguish obesity, prediabetes, type 2 diabetes, and type 1 diabetes wherever individual studies are discussed. The islet-directed subsection now separates evidence from experimental β-cell injury models, type 2 diabetes, and autoimmune diabetes and emphasizes the different therapeutic questions and endpoints relevant to each setting. The translational barriers section also clarifies that prediabetes represents an earlier dysglycemic state and should be interpreted within that specific context. These revisions improve disease-specific interpretation while preserving the mechanistic organization of the Review.

Comment 16.

Lines 506–536: For hepatic GABA-targeted strategies, the authors should specify measurable biomarkers of pathway engagement, such as hepatic GABA-T activity, hepatic GABA abundance, vagal readouts, fasting insulin, or insulin sensitivity.

Response: We appreciate this suggestion. Section 5.2 now distinguishes biomarkers of pathway engagement from downstream metabolic outcomes. Hepatic GABA-transaminase activity and hepatic GABA abundance are identified as measures of hepatic target engagement, while hepatic GABA release, hepatocyte membrane potential, and afferent vagal activity are presented as indicators of the liver–neural pathway. Fasting insulin, HOMA-IR, clamp-derived insulin sensitivity, feeding behavior, and peripheral glucose disposal are discussed as complementary systemic outcomes. The revised text also emphasizes that changes in circulating GABA or whole-body metabolism alone do not demonstrate selective engagement of the obesity-associated hepatic GABA pathway.

Comment 17.

Lines 537–580: The adipose–immune–microbiota discussion needs better consideration of confounding factors. Changes in food intake, body weight, microbiota composition, and inflammatory tone may all influence the reported outcomes.

Response: We revised Sections 5.3 and 5.4 to address this concern. Section 5.3 now presents the experimental evidence within an integrated adipose–immune–microbiota framework and distinguishes GABA-associated effects on adipose remodeling, immune and stromal-cell activity, microbial composition, and depot-specific responses. Section 5.4 now explicitly discusses potential confounding factors, including changes in food intake, body weight, microbiota composition, intestinal barrier function, microbial inflammatory products, and inflammatory tone. The revised discussion distinguishes direct GABA-sensitive mechanisms from indirect effects mediated through energy balance, adiposity, microbial ecology, and inflammation and emphasizes the need for tissue-specific, mechanism-matched evidence of pathway engagement.

Comment 18.

Lines 581–624 and Box 1: The limitations of the current evidence should be stated more directly. In particular, the review should acknowledge the heavy reliance on animal/cell-based evidence, limited human validation, and uncertainty about active GABA sources.

Response: We revised Section 5.4 to state these limitations more explicitly. The revised section now clarifies that much of the mechanistic evidence derives from animal and cell-based studies, whereas human validation remains limited. We also expanded the discussion of uncertainty regarding the active source of GABA and emphasize that dietary, microbial, and endogenous sources cannot yet be linked unequivocally to tissue-specific pathway engagement. These limitations are further highlighted in the revised Box 1 through additional unresolved translational questions.

Comment 19.

Lines 651–850: The reference format is inconsistent. Please standardize author names, journal titles, capitalization, punctuation, DOI style, and spacing according to the journal guidelines.

Response: We thank the reviewer for identifying these inconsistencies. The reference list has been thoroughly reviewed and standardized according to IJMS formatting requirements, including author names, journal titles, capitalization, punctuation, spacing, and DOI presentation. The references were also checked for internal consistency and accuracy.

Comment 20.

Page 1 around Line 43 and the back matter: Several template elements appear incomplete, including editorial placeholders and publication statements. The authors should remove placeholders and complete the funding, conflict-of-interest, data availability, and related declarations before submission.

Response: We appreciate the reviewer for identifying these omissions. All remaining template placeholders and editorial statements have been removed or completed. The funding statement, conflicts of interest, data availability statement, institutional review board statement, informed consent statement, acknowledgments, and other required declarations have been reviewed and updated, where applicable, in accordance with IJMS submission requirements.

Comment 21.

Lines 20–38, 72–79, 326–339, and 625–645: Most importantly, the review lacks a clear conceptual backbone. The authors repeatedly mention adaptive, compensatory, and maladaptive GABA signaling, but do not develop these terms into their own theoretical model. As a result, the manuscript reads more like a collection of tissue-specific observations than a thesis-driven review. The authors should define a core framework, for example linking GABA source, tissue access, target cell/receptor, disease state, and functional output, and then use this framework consistently throughout the manuscript.

Response: We sincerely thank the reviewer for this insightful recommendation. The manuscript has been substantially reorganized around five connected determinants of peripheral GABA function: GABA source, tissue access, responding cell or receptor pathway, disease state, and functional metabolic outcome. This framework is introduced in the Abstract and Introduction. Adaptive signaling is defined as a response that preserves tissue regulation, compensatory signaling as a response that limits metabolic stress without fully restoring homeostasis, and maladaptive signaling as a response that reinforces disease-associated dysfunction. The framework is then applied throughout the Review by distinguishing intracellular GABA metabolism from extracellular receptor signaling and systemic exposure, separating host-derived, dietary, and microbiota-derived sources, and interpreting tissue-specific effects according to the responding cell population and physiological context. The revised disease-remodeling section and Figure 3 further show how obesity and type 2 diabetes alter the direction of GABA-sensitive responses. The translational section and Box 1 extend the same framework to pathway engagement, therapeutic selectivity, human validation, and unresolved questions. Together, these revisions provide a consistent conceptual backbone and integrate the tissue-specific evidence into a unified interpretation of peripheral GABA signaling in metabolic adaptation and maladaptation.

Reviewer 2 Report

Comments and Suggestions for Authors

This is a timely and generally well-written review with a useful and interesting framing of peripheral GABA signalling as context-dependent. The manuscript covers the topic broadly and should be of interest to readers in the field. I have only a few suggestions for revision.

The distinction between adaptation, compensation, and maladaptation could be defined more clearly early in the manuscript and then applied more consistently across the later sections. The discussion of peripheral GABA receptor biology would also benefit from a little more precision, particularly with respect to GABAA receptor subunit composition in different tissues, which may help explain why GABA can be depolarising in some settings but not others.

A little more specificity would also strengthen the liver and adipose sections. For example, it would be helpful to comment on whether the obesity-associated increase in hepatic GABA is thought to be mainly host-derived or whether microbial GABA might also contribute via the portal circulation, and to clarify which adipose cell populations currently have the best evidence for functional GABA receptor expression. The translational section would also benefit from a brief comment on possible reasons for the limited clinical translation of encouraging preclinical findings.

A few minor points should also be addressed. The wording in lines 47–49 on page 2 is somewhat repetitive (“extra-neural compartments” and “outside the nervous system”). Figure 1 would benefit from a higher-resolution version. On page 3, lines 110–111, please check whether GAD1 and GAD2 should be italicised if gene symbols are intended.

Overall, I think the manuscript would be suitable for publication after minor revision.

Author Response

Response to Reviewer 1

We sincerely thank the reviewer for the positive evaluation of our manuscript and for the constructive suggestions. We have carefully revised the manuscript to improve conceptual clarity, mechanistic precision and translational interpretation. Our responses are provided below.

Comment 1.

The distinction between adaptation, compensation, and maladaptation could be defined more clearly early in the manuscript and then applied more consistently across the later sections.

Response

We appreciate this suggestion. We revised both the Abstract and Introduction to define adaptive regulation, compensatory remodeling and maladaptive signaling more explicitly at the outset of the review. These concepts now provide the organizational framework for interpreting peripheral GABA signaling throughout the manuscript and are applied consistently when discussing physiological regulation, obesity-associated tissue remodeling and translational opportunities.

Comment 2.

The discussion of peripheral GABA receptor biology would also benefit from a little more precision, particularly with respect to GABAA receptor subunit composition in different tissues, which may help explain why GABA can be depolarizing in some settings but not others.

Response

Thank you for this helpful suggestion. We expanded Section 2.2 to provide a more detailed discussion of peripheral GABA receptor biology. The revised text now explains that GABAA_AA-receptor responses depend on receptor subunit composition, extracellular GABA concentration, chloride gradients and cation–chloride cotransporter activity. We further distinguish the contrasting physiological responses observed in pancreatic α-cells and human β-cells, illustrating how cell-specific chloride homeostasis determines whether GABAA_AA-receptor activation produces membrane hyperpolarization or depolarization.

Comment 3.

Response

We appreciate this suggestion and revised both sections accordingly. In the liver section, we now distinguish the obesity-associated hepatic GABA-output pathway from systemic GABA exposure and explicitly note that the experimental evidence supporting the hepatic vagal pathway is based on host-derived hepatocyte GABA production. We also acknowledge that whether microbiota-derived GABA contributes to hepatic GABA signaling through the portal circulation remains unresolved. In the adipose section, we reorganized the discussion around coordinated adipocyte, immune and stromal remodeling rather than isolated cellular observations and clarify that the strongest functional evidence currently involves coordinated responses across adipocytes, macrophages, stromal cells and the gut microbiota, while recognizing that the directly responding cell populations and receptor-dependent mechanisms remain incompletely defined.

Comment 4.

The translational section would also benefit from a brief comment on possible reasons for the limited clinical translation of encouraging preclinical findings.

Response

Thank you for this valuable recommendation. We substantially expanded the translational section to address the major barriers limiting clinical translation. The revised discussion emphasizes that peripheral GABA abundance alone does not establish pathway engagement, that therapeutic responses are highly tissue- and disease-stage dependent, and that successful translation will require pathway-specific biomarkers, evidence of target engagement and mechanism-matched clinical endpoints. These concepts are further synthesized in the revised Conclusions and Outlook section.

Comment 5.

The wording in lines 47–49 on page 2 is somewhat repetitive (“extra-neural compartments” and “outside the nervous system”).

Response

We revised the Introduction to remove this redundancy and improve readability.

Comment 6.

Figure 1 would benefit from a higher-resolution version.

Response

A higher-resolution version of Figure 1 has been prepared and will be provided in the revised submission.

Comment 7.

On page 3, lines 110–111, please check whether GAD1 and GAD2 should be italicized if gene symbols are intended.

Response: We thank the reviewer for identifying this formatting issue. The gene symbols GAD1 and GAD2 have now been italicized, whereas the corresponding protein isoforms GAD67 and GAD65 remain in roman type.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript (Review) by Saliu et al. «Peripheral GABA Signaling in Metabolic Adaptation and Maladaptation» is important because provides complex information about the biochemical basis of peripheral GABA Signaling, GABA-sensitive pathways, their organisation, that may provide a basis for more precise therapeutic approaches in metabolic disease. In the introduction, the authors clearly formulate the initial reasons for writing the review and then gradually reveal the topic and achieve the aim. the article is important not only for fundamental but also for applied science. Very good, clear illustrations. The text is structured consistently, clearly and understandably. I have no comments for corrections for this article.

Author Response

Response to Reviewer 3

Response:
We sincerely thank the reviewer for the careful evaluation of our manuscript and for the positive comments. We greatly appreciate the reviewer's recognition of the conceptual framework, organization, and translational relevance of our review, as well as the clarity of the figures and presentation. We are encouraged that the reviewer found the manuscript to be a valuable contribution to both fundamental and applied research on peripheral GABA signaling. We appreciate the reviewer's recommendation for publication and thank them for their thoughtful assessment.

Reviewer 4 Report

Comments and Suggestions for Authors

Comment 1. Islet physiology requires mechanistic clarification

Section 3.1 (Lines 228–233): The description of VIAAT and GAT3 distribution is largely descriptive and lacks sufficient functional context. To make this review more self-contained and informative, the authors should explicitly explain the physiological sequence linking β-cell GABA release to islet hormone regulation. In particular, please clarify how GABA is co-released with insulin and how cell-specific chloride gradients determine whether GABA_A receptor activation induces membrane hyperpolarization or depolarization in α- and β-cells, thereby regulating glucagon and insulin secretion.

Comment 2. The hepatic GABA–HVAN axis should be described mechanistically

Section 3.2 (Lines 251–253): The discussion of hepatic GABA remains mechanistically underdeveloped. Because the hepatic vagal afferent nerve (HVAN) axis represents one of the best-characterized pathways linking peripheral GABA signaling to systemic metabolism, the authors should provide a more comprehensive description of the proposed mechanistic cascade. Specifically, the discussion should include the progression from hepatic steatosis to hepatocyte membrane depolarization, Na+-coupled GABA export and GABA-transaminase (GABA-T) upregulation, activation of GABA_A receptors on hepatic vagal afferent neurons, suppression of HVAN firing, and the subsequent development of hyperinsulinemia and impaired skeletal muscle glucose uptake. Although Section 4 briefly revisits this pathway, integrating these mechanistic concepts into the physiology section would substantially improve continuity and reader comprehension.

Comment 3. The adipose tissue and immune sections require stronger mechanistic integration

Sections 3.2–3.4 and 4.3: The discussions of adipose tissue remodeling and peripheral immune regulation remain largely descriptive and rely heavily on associative statements rather than mechanistic interpretation. Instead of listing individual observations, the review should integrate these findings within the framework of obesity-associated metabolic inflammation (metainflammation).

Specifically, the authors should discuss how GABAergic signaling in macrophages, T cells, stromal cells, and adipocytes contributes to inflammatory remodeling of adipose tissue and/or the liver, and how these interactions ultimately influence systemic insulin sensitivity, glucose homeostasis, or energy expenditure.

Furthermore, the proposed gut–adipose axis requires substantially greater mechanistic detail. If supported by reference [36], the authors should specify (i) the bacterial taxa altered by GABA treatment, (ii) the candidate gut-derived mediators (e.g., microbial GABA, short-chain fatty acids, or bile acids), and (iii) the immune or stromal cell populations responsible for initiating white-to-beige adipose remodeling. Without defining these cellular and molecular components, the discussion remains largely phenomenological.

Comment 4. The overall organization should better integrate physiology and pathophysiology

Overall manuscript organization: While Section 4 provides substantially greater mechanistic depth than Section 3, separating physiological regulation and disease-associated maladaptation into completely independent sections creates a noticeable structural disconnect. Readers encounter simplified physiological descriptions in Section 3, whereas many of the mechanistic details are introduced only later in the disease context.

The manuscript would be substantially strengthened by organizing each organ system (pancreatic islets, liver, adipose tissue, immune cells, and skeletal muscle) into integrated "physiology versus pathology" subsections, or at minimum by providing stronger cross-referencing between Sections 3 and 4. This organization would improve readability, reduce redundancy, and allow readers to directly compare normal and disease-associated GABA signaling within the same biological context.

For unresolved topics, particularly adipose tissue remodeling, a schematic figure or comparative table summarizing the proposed host-derived versus microbiota-derived GABA pathways, their candidate mediators, and their potential target cell populations would also improve the manuscript and provide a clearer framework for future research.

Author Response

Response to Reviewer 4

We sincerely thank the reviewer for the thoughtful and constructive comments. We have carefully revised the manuscript to strengthen the mechanistic interpretation of peripheral GABA signaling, improve continuity between physiological and disease-associated pathways, and clarify the translational framework. All suggested sections have been substantially revised, as detailed below.

Comment 1. Islet physiology requires mechanistic clarification.

Response:

We appreciate this suggestion and have substantially expanded Section 3.1 to provide a more mechanistic description of intra-islet GABA signaling. The revised text now explains that β-cell-derived GABA is released during glucose-stimulated β-cell activity, discusses the relationship between GABA release and insulin secretion, and clarifies that these signals may originate from distinct intracellular pools. We further describe how cell-specific chloride gradients determine the functional consequences of GABAA_AA-receptor activation, leading to membrane hyperpolarization and glucagon suppression in α-cells, but membrane depolarization and enhanced excitability in human β-cells. Finally, we distinguish the evidence supporting glucagon regulation, β-cell secretory modulation, and β-cell proliferation to provide a clearer mechanistic framework for islet physiology.

Comment 2. The hepatic GABA–HVAN axis should be described mechanistically.

Response:

We thank the reviewer for this valuable recommendation. Section 3.2 has been substantially revised to provide a more complete physiological description of the hepatic GABA pathway. The revised section now explains how hepatocyte membrane potential regulates hepatic GABA release and how this influences hepatic vagal afferent signaling before introducing the disease-associated amplification of this pathway in obesity. We also expanded Section 4.2 to describe obesity-induced hepatocyte depolarization, increased GABA-transaminase-dependent GABA production, suppression of hepatic vagal afferent activity, and the downstream consequences for hyperinsulinemia, insulin resistance, and skeletal muscle glucose disposal. Together, these revisions improve continuity between normal physiology and disease-associated remodeling while avoiding unnecessary repetition between Sections 3 and 4.

Comment 3. The adipose tissue and immune sections require stronger mechanistic integration.

Response:

We appreciate this important suggestion and substantially revised both the physiological and disease-remodeling sections. Section 4.3 has been reorganized around obesity-associated tissue remodeling rather than individual experimental observations. The revised discussion now integrates adipocytes, macrophages, stromal cells, and gut microbiota within the context of coordinated immune–metabolic remodeling and emphasizes how GABA-associated responses emerge from interactions among multiple cellular compartments rather than a single adipocyte-specific pathway. We also expanded the discussion of the gut–adipose axis by describing the bacterial taxa altered following GABA treatment, the fecal microbiota transfer experiments supporting microbiota involvement, and the resulting effects on adipose thermogenic remodeling. Throughout the revision, we emphasize the current mechanistic evidence while clearly distinguishing experimentally demonstrated findings from areas that remain unresolved.

Comment 4. The overall organization should better integrate physiology and pathophysiology.

Response:

We appreciate the reviewer's thoughtful recommendation regarding manuscript organization. Rather than completely restructuring the review into organ-specific physiology–pathology subsections, we strengthened the conceptual continuity between Sections 3 and 4 while preserving the overall organization of the manuscript. Section 3 now provides substantially greater mechanistic detail for normal physiological pathways, whereas Section 4 focuses on how obesity and type 2 diabetes remodel those same pathways into adaptive, compensatory, or maladaptive responses. This approach reduces redundancy while allowing readers to distinguish physiological mechanisms from disease-associated remodeling more clearly. In addition, we developed a new disease-remodeling schematic (Figure 3) that summarizes the major tissue-specific changes occurring in pancreatic islets, liver, and adipose tissue during metabolic disease and highlights the context-dependent nature of peripheral GABA signaling.

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The revised manuscript has improved considerably and addresses most of my previous comments. The additional discussion on the mechanisms is helpful. However, a few minor points still need clarification.

Islet GABA signaling

The revised explanation of β-cell GABA release, the chloride gradients in α- and β-cells, and the effects of GABA-A receptor activation is clear enough. The distinction between findings from rodent and human islets is also appropriate.

Hepatic GABA–HVAN axis

The explanation of the pathway from hepatocyte depolarization to increased hepatic GABA production and release, reduced hepatic vagal afferent nerve activity, hyperinsulinemia, and impaired glucose disposal is much clearer than before. However, the roles of the transporters and receptors are still not well explained. Please briefly describe these steps, or clearly state that the detailed mechanisms are still not fully understood.

Adipose–immune–microbiota axis

This section has improved with the addition of adipose inflammation, stromal cell changes, macrophage infiltration, altered gut microbiota, and fecal microbiota transfer studies. However, the causal relationship is still not clear. Statements suggesting that specific microbiota changes directly cause adipose tissue remodeling should be more careful unless the responsible bacterial taxa, metabolites, and target host cells have been clearly identified. It would also be helpful to distinguish more clearly between findings supported by evidence and proposed mechanisms.

Organization

The transition between the physiology and disease-related sections is still a little abrupt. A major reorganization is not necessary, but adding a few cross-references between the corresponding physiology and pathology sections would make the manuscript easier to follow.

Author Response

Islet GABA signaling

Reviewer comment:
The revised explanation of β-cell GABA release, the chloride gradients in α- and β-cells, and the effects of GABA(_A)-receptor activation is clear enough. The distinction between findings from rodent and human islets is also appropriate.

Response:
We thank the reviewer for this positive assessment. No further changes were required in response to this comment.

Hepatic GABA signaling

Reviewer comment:
The explanation of the pathway from hepatocyte depolarization to increased hepatic GABA production and release, reduced hepatic vagal afferent nerve activity, hyperinsulinemia, and impaired glucose disposal is much clearer than before. However, the roles of the transporters and receptors are still not well explained. Please briefly describe these steps, or clearly state that the detailed mechanisms are still not fully understood.

Response:
We thank the reviewer for this important comment. We have revised Section 3.2 to clarify the evidence concerning hepatic GABA transport and receptor engagement. The revised text explains that extracellular hepatic GABA availability is influenced by hepatocyte membrane potential and electrogenic solute carrier 6 transporters. BGT1/SLC6A12 and GAT2/SLC6A13 appear to function predominantly in GABA uptake, whereas TauT/SLC6A6 and CRT/SLC6A8 have been proposed as possible routes of GABA efflux. We now state explicitly that the transporter responsible for hepatocyte GABA export has not been definitively identified.

We have also clarified that extracellular GABA suppresses hepatic vagal afferent firing and that the ability of the GABA_A-receptor agonist muscimol to reproduce this effect supports a GABA_A-receptor-sensitive step in the pathway. However, the relevant receptor subunits, the receptor-bearing cell population, whether GABA acts directly on vagal afferent endings, and the downstream neural circuitry remain unresolved. The revised text therefore distinguishes the experimentally supported steps from the mechanisms that remain to be established.

These revisions appear in Section 3.2, page 11, lines 325–357.

Adipose–immune–microbiota axis

Reviewer comment:
This section has improved with the addition of adipose inflammation, stromal cell changes, macrophage infiltration, altered gut microbiota, and fecal microbiota transfer studies. However, the causal relationship is still not clear. Statements suggesting that specific microbiota changes directly cause adipose tissue remodeling should be more careful unless the responsible bacterial taxa, metabolites, and target host cells have been clearly identified. It would also be helpful to distinguish more clearly between findings supported by evidence and proposed mechanisms.

Response:
We thank the reviewer for this important clarification. We have revised Section 4.3 to distinguish more clearly between the functional evidence provided by fecal microbiota transfer and the molecular mechanisms that remain unresolved. The revised text states that oral GABA alters microbial community composition in obese mice and that transfer of fecal microbiota from GABA-treated donors increases thermogenic gene expression in the inguinal white adipose tissue of recipient mice [43]. We interpret this finding as evidence for a transferable microbiota-associated component of the adipose response.

We now state explicitly that these experiments do not demonstrate that individual bacterial taxa altered by GABA treatment directly cause adipose remodeling, nor do they establish that GABA itself is the transferred mediator. The responsible microbial functions or metabolites, their route of communication with adipose tissue, and the host target cells remain unidentified. The proposed microbiota–adipose relationship is therefore presented as an emerging model rather than an established taxon–metabolite–host-cell pathway.

Section 5.3 has also been revised so that microbiota-associated signaling is presented as a potential indirect route for modulating white-adipose plasticity. This incompletely resolved pathway is distinguished from the more direct evidence that GABA_B-receptor signaling impairs brown-adipose thermogenic function during obesity [30].

These revisions appear in Sections 4.3 and 5.3, pages 16 & 18, lines 533–577 & Line 655-679.

Organization

Reviewer comment:
The transition between the physiology and disease-related sections is still a little abrupt. A major reorganization is not necessary, but adding a few cross-references between the corresponding physiology and pathology sections would make the manuscript easier to follow.

Response:
We thank the reviewer for this helpful suggestion. We have revised the opening of Section 4 to provide a clearer transition from the organ-specific physiological discussion in Section 3 to the disease-associated remodeling examined in Section 4. We have also added selective cross-references in Sections 4.1 and 4.2 to the corresponding discussions of intra-islet GABA signaling in Section 3.1 and hepatic GABA–vagal signaling in Section 3.2, respectively. These revisions improve continuity without changing the overall organization or duplicating content.

The revisions appear in Sections 4, 4.1, and 4.2, pages 13–15, lines 428–513.