Hepatic HKDC1 Deletion Alleviates Western Diet-Induced MASH in Mice
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Abstract
The global prevalence of Metabolic dysfunction-associated steatohepatitis (MASH) has been rising sharply, closely mirroring the increasing rates of obesity and metabolic syndrome. MASH exhibits a strong sexual dimorphism where females are affected with more severe forms after menopause. Hexokinase domain-containing protein 1 (HKDC1) has recently been recognized for its role in liver diseases, where its expression is minimal under normal conditions but significantly increases in response to metabolic stressors like obesity and liver injury. This selective upregulation suggests HKDC1’s potential specialization in hepatic glucose and lipid dysregulation, linking it closely to the progression of MASLD and MASH. This study aims to clarify the role of HKDC1 in Western diet-induced MASH in female mice by examining its impact on hepatic glucose and lipid metabolism, offering insights into its potential as a therapeutic target and addressing the need for sex-specific research in liver disease. This study reveals that HKDC1 expression is elevated in obese women with MASH and correlates with liver pathology. In a mouse model, liver-specific HKDC1 knockout (HKDC1 LKO ) protected against Western diet-induced obesity, glucose intolerance, and MASH features, including steatosis, inflammation, and fibrosis. Transcriptomic analysis showed that HKDC1 deletion reduced pro-inflammatory and pro-fibrotic gene expression, while gut microbiome analysis indicated a shift toward MASH-protective bacteria. These findings suggest that HKDC1 may exacerbate MASH progression through its role in metabolic and inflammatory pathways, making it a potential therapeutic target.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14886749.
"Hepatic HKDC1 Deletion Alleviates Western Diet-Induced MASH in Mice" investigates the role of hexokinase domain containing 1 (HKDC1) in the development of metabolic dysfunction-associated steatohepatitis (MASH) induced by a Western diet in mice. The study demonstrates that deletion of HKDC1 in hepatic tissues leads to a significant reduction in liver inflammation, steatosis, and fibrosis associated with MASH i.e reduced pro-inflammatory and pro-fibrotic gene expression, while gut microbiome analysis indicated a shift toward MASH-protective bacteria. This research contributes to the field by elucidating a potential therapeutic target for treating metabolic liver diseases, particularly …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14886749.
"Hepatic HKDC1 Deletion Alleviates Western Diet-Induced MASH in Mice" investigates the role of hexokinase domain containing 1 (HKDC1) in the development of metabolic dysfunction-associated steatohepatitis (MASH) induced by a Western diet in mice. The study demonstrates that deletion of HKDC1 in hepatic tissues leads to a significant reduction in liver inflammation, steatosis, and fibrosis associated with MASH i.e reduced pro-inflammatory and pro-fibrotic gene expression, while gut microbiome analysis indicated a shift toward MASH-protective bacteria. This research contributes to the field by elucidating a potential therapeutic target for treating metabolic liver diseases, particularly those exacerbated by dietary factors. By focusing on HKDC1, the authors provide insights into metabolic pathways that could be manipulated to mitigate liver damage and improve overall metabolic health.
Major issues
Additional study may be required to validate these findings in larger animal models or clinical trials involving human subjects.
There is lack of exploration into the underlying mechanisms by which HKDC1 deletion exerts its protective effects against MASH. A more detailed investigation into signaling pathways and metabolic changes resulting from HKDC1 loss will strengthen the conclusions drawn from this study.
Minor issues
Some figures could benefit from clearer labeling and legends to enhance understanding for readers unfamiliar with specific methodologies used.
Authors provide more context on how dietary components specifically influence HKDC1 expression will improve flow and coherence within the manuscript.
The discussion section will be more enhanced by including more references to existing literature on similar pathways involved in liver metabolism and disease progression.
Competing interests
The author declares that they have no competing interests.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14782675.
This review incorporates comments and contributions from Femi Arogundade, Randa Salah Gomaa Mahmoud, Teena Bajaj, Chen Yang, Faith Onditi, and Gliday Yuka. This review was synthesized by Femi Arogundade.
Summary of the Study:
The study investigates the role of Hexokinase domain-containing protein 1 (HKDC1) in Metabolic dysfunction-associated steatohepatitis (MASH), focusing on female mice. They found that HKDC1 levels were elevated in MASH patients and correlated with disease severity. Using a liver-specific HKDC1 knockout (HKDC1LKO) mouse model, they demonstrated that HKDC1 deletion protected mice from obesity, glucose intolerance, liver damage, and inflammation. The findings suggest that …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/14782675.
This review incorporates comments and contributions from Femi Arogundade, Randa Salah Gomaa Mahmoud, Teena Bajaj, Chen Yang, Faith Onditi, and Gliday Yuka. This review was synthesized by Femi Arogundade.
Summary of the Study:
The study investigates the role of Hexokinase domain-containing protein 1 (HKDC1) in Metabolic dysfunction-associated steatohepatitis (MASH), focusing on female mice. They found that HKDC1 levels were elevated in MASH patients and correlated with disease severity. Using a liver-specific HKDC1 knockout (HKDC1LKO) mouse model, they demonstrated that HKDC1 deletion protected mice from obesity, glucose intolerance, liver damage, and inflammation. The findings suggest that HKDC1 influences inflammation, metabolism, and gut health, making it a potential therapeutic target for MASH, particularly in women.
Positive Aspects of the Study:
The study provides new insights into HKDC1's role in MASH, particularly in female mice.
Findings suggest HKDC1 as a potential therapeutic target, which could help in developing new treatments for MASH.
The use of liver-specific HKDC1 knockout (HKDC1LKO) mice allows for good mechanistic insights into HKDC1's role in disease progression.
The study examines multiple aspects of MASH, including metabolic function, inflammation, and gut microbiome.
Major Comments:
The materials and methods section needs to be rearranged so all the animal procedures are put together and the human procedures are presented in one section.
The materials and methods section should be reorganized to clearly describe what samples were collected, when and how they were collected, and what assays were conducted for each of these samples.
The study focused only on female mice, making it impossible to draw direct sex-specific comparisons.
The animal experiment section states: "When mice reached 8 weeks of age, Western diet (Research diet, D12079Bi, NJ, USA) or normal chow (Envigo-7912, IN, USA) were given for 28 weeks." However, study design in Figure 1 shows 20 weeks. Clarify how long the mice were fed on either diet.
The composition of the Western diet should be described in detail.
The statement: "Glucose (2mg glucose ip/g) and pyruvate (2mg sodium pyruvate ip/g) tolerance tests were performed in overnight fasted mice." The results of this test were not illustrated; consider adding relevant data.
In the Body Composition, Glucose Homeostasis, and Metabolic Rate section, samples used for conducting the tests should be clearly described (e.g., how much blood was obtained from the lateral vein?).
Specify whether each mouse underwent all tests sequentially (overnight fasting → glucose/pyruvate tolerance test → continued fasting for 4 hours for insulin tolerance test).
Explain AUC (Area Under the Curve) in Figures 4B and 4D.
While gut microbiome changes were observed, causality was not fully established.
Provide clarity regarding food access. The first paragraph of the Methods section states that the mice had ad libitum access to food, implying unrestricted feeding. However, the Food Intake section describes the use of BioDAQ cages for monitoring food intake, along with an acclimatization period. Clarify whether food remained available ad libitum during the measurement period and whether the acclimatization phase involved any temporary restrictions.
Details of histopathology examination were not mentioned in the Materials and Methods section.
Minor Comments:
Title Revision: The study design includes both human and animal protocols; however, the title only mentions animals without specifying gender. The title should be revised to reflect both aspects. Also, identify the abbreviation in the title as Hexokinase domain-containing protein 1 (HKDC1) and Metabolic dysfunction-associated steatohepatitis (MASH).
In the Abstract section, include a clear summary of methods.
The hypothesis should be clearly stated in the Abstract.
Identify MASLD as Metabolic Dysfunction-Associated Steatotic Liver Disease in the Abstract.
In the introduction the statement "The rise in mortality rates for females, from 0.2 to 2 per 100,000, with an average annual percent change (AAPC) of 11.7% (p<0.001), compared to the rise in males, from 0.2 to 1.3 per 100,000 with an AAPC of 9.3% (p<0.001)" should be restructured. If p-value (p<0.001) indicates a significant difference between males and females, it should be mentioned once at the end of the comparison sentence.
In the introduction, the statement "Current estimates suggest that up to 25% of the global population may be affected by MASLD…" should include mortality rates and statistics on MASH-related complications.
In the introduction the statement "While MASH tends to initially affect younger males more frequently, it's important to recognize that females over 60 experience a higher prevalence of MASH," specify the age range for "younger males" and revise "females over 60" to "females over 60 years."
In the Anthropometric and Biochemical Measurements section, the statement: "Preoperative clinical data were collected on the day of the surgery after an overnight fast of at least 12 hours and serum was separated by centrifugation." should be revised to replace clinical data with blood samples for clarity.
Provide key references in the introduction for the following statements/facts:
"Central to the development of MASH is the dysregulation of both glucose and lipid metabolism…………. and subsequent fibrogenesis "
"Unlike other members of the hexokinase family, which are ubiquitously expressed and primarily function in glycolysis……………… and lipid metabolism further underscores its potential significance in MASH pathogenesis"
Identify NEFA as Non-Esterified Fatty Acid.
Identify GSEA as Gene Set Enrichment Analysis.
In the Discussion section:
The statement: "MASH has become a health concern as its global prevalence rate is 30%" - The 30% prevalence refers to MASLD, not MASH. Cite recent data separately for both.
"Strong positive correlation" needs actual r-values and p-values. The phrase "strong positive correlation" should be supported with r-values and p-values (e.g., "Our human cohort data established a strong positive correlation (r=..., p=...) between hepatic HKDC1 expression and MASLD progression in females"). Provide specific statistical values.
The statement: "Notably, our female mice (~36 weeks of age at the end of the experimental period) would not have reflected menopausal phenotype, again underscoring the nutrient excess-HKDC1 expression link." Consider citing literature on modelling menopause in rodents and clarifying the claim.
Comments on Reporting (Information on the Statistical Analyses or Availability of Data):
Statistical Methodology Issues:
Authors state "means ± standard error of deviation (SD)," which is incorrect. It should be either standard error (SE) or standard deviation (SD).
No justification provided for choice between Tukey vs. Bonferroni post-hoc tests.
No mention of testing for homogeneity of variance before conducting ANOVA.
Lack of details on normality assessment for small sample sizes.
Missing Information:
No description of how outliers were identified and handled.
No mention of sample size calculations or power analysis.
No specifics about RNA-seq analysis parameters (e.g., FDR correction method).
No details about metabolomics data preprocessing and normalization.
Reporting Gaps:
No mention of effect sizes.
No information about which specific tests were used for which comparisons.
No statement about data availability.
Identify DEG as Differentially expressed genes.
Suggestions for Future Studies:
Investigate the relationship between HKDC1 and cholesterol/bile acid metabolism more deeply to strengthen mechanistic conclusions.
Investigate HKDC1 expression in male MASH models and compare with females to determine sex-specific effects.
Conduct studies on postmenopausal women to assess hormonal influence on HKDC1-mediated MASH progression.
Competing interests
The authors declare that they have no competing interests.
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In summary, the gut microbiome of HKDC1 liver-specific knockout mice has fewer bacterial strains that positively correlate with MASH, which may partially contribute to or result from the improved phenotype of mice with hepatic HKDC1 ablation.
Very interesting that HKDC1 KO reduces uptake of lipids from the gut. Are you able to experimentally determine whether there is a link between changes in microbiome composition and lipid update? How would you do this?
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Gene ontology analysis of the downregulated genes revealed that the “extracellular matrix organization” pathway is the most affected pathway (Figure 7C). A further in-depth analysis of this pathway revealed that most of the genes belonged to the pro-fibrogenic program (Fig 7D)
How does HKDC1 ablation reduce fibrotic signaling?
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a possible mechanism by which HKDC1 upregulation exacerbates MASH development is through dysregulating cholesterol metabolism.
Do you have a hypothesis as to how HKDC1 contributes to dysregulated cholesterol metabolism?
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More importantly, HKDC1 levels were not only significantly upregulated in differentiated HLO with respect to undifferentiated HLO but were also increased after PA treatment (Fig 1H), suggesting that a high lipid environment can regulate HKDC1 expression.
Why does treatment with albumin also lead to upregulation of HKDC1 in your organoid experiment?
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