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Cited by 357Gut microbiota and healthLiver Disease Diagnosis and TreatmentDiet, Metabolism, and Disease

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Gut Akkermansia muciniphila ameliorates metabolic dysfunction-associated fatty liver disease by regulating the metabolism of L-aspartate via gut-liver axis

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Rao, Yong, Kuang, Zhiqi, Li, Chan, Guo, Shiyao, Xu, Yaohao, Zhao, Dandan, Hu, Yutao, Song, Bingbing, Jiang, Zhi, Ge, Zhenhuang, Liu, Xiyuan, Li, Chengdao, Chen, Shuobin, Ye, Jiming, Huang, Zhishu, Lu, Yongjun·2021·Gut Microbes
DOI 10.1080/19490976.2021.1927633PMC PMC8158032
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Abstract

The gut bacterium Akkermansia muciniphila has been increasingly recognized for its therapeutic potential in treating metabolic disorders, including obesity, diabetes, and metabolicdysfunction-associated fatty liver disease (MAFLD). However, its underlying mechanism involved in its well-known metabolic actions needs further evaluation. The present study explored the therapeutic effect and mechanism of A. muciniphila in intervening MAFLD by using a high-fat and high-cholesterol (HFC) diet induced obese mice model. Mice treated with A. muciniphila efficiently reversed MAFLD in the liver, such as hepatic steatosis, inflammatory, and liver injury. These therapeutic effects persisted after long-term drug withdrawal and were slightly weakened in the antibiotics-treated obese mice. A. muciniphila treatment efficiently increased mitochondrial oxidation and bile acid metabolism in the gut-liver axis, ameliorated oxidative stress-induced cell apoptosis in gut, leading to the reshaping of the gut microbiota composition. These metabolic improvements occurred with increased L-aspartate levels in the liver that transported from the gut. The administration of L-aspartate in vitro or in mice displayed the similar beneficial metabolic effects mentioned above and efficiently ameliorated MAFLD. Together, these data indicate that the anti-MAFLD activity of A. muciniphila correlated with lipid oxidation and improved gut–liver interactions through regulating the metabolism of L-aspartate. A. muciniphila could be a potential agent for clinical intervention in MAFLD.

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Figure 1. A. muciniphila attenuated obese MAFLD induced by HFC diet feeding. (a-h) Eight-week-old male C57BL/6 mice were fed with an HFC diet for 11 weeks to induce obese MAFLD. They were then randomly divided into two subgroups to treat with PBS as a control (HFC group) or A. muciniphila in PBS (HFC+A. muciniphila) orally every other day for 6 weeks. (a) Schematic diagram of A. muciniphila treatment. (b) Abundance of A. muciniphila in feces. (c) Mouse body weight at week 17. (d-e) Hepatic triglyceride (TG) and diacylglyceride (DAG) levels. (f) Plasma levels of AST, ALT and ALP. (g) Pathologic examination of the liver by hematoxylin ＆ eosin (H & E) staining, oil red O staining (ORO), and immunofluorescence examination of IL-6 and CD68. Representative images were captured. Scale bar, 200 μm. (h) Expression of mRNA markers for steatosis and inflammation markers. The levels of genes in the HFC control group were set as 1, and the relative fold increases were determined by comparison with the HFC control group. N = 5–8 mice/group. * p < .05, compared with HFC control group mice. (i-m) Sustainability of the anti-MAFLD efficacy of A. muciniphila after the cessation of its administration. (i) Schematic diagram of the experimental design. After 6 weeks of administration, A. muciniphila was withdrawn from the HFC-induced obese MAFLD mice followed by a period of 4 weeks of washout to evaluate of the sustainability of the anti-MAFLD efficacy. (j) Mouse body weight. (k) TG level in the liver. (l) Plasma levels of AST, ALT and ALP. (m) Pathologic examination of the liver by H ＆ E staining, oil red O staining, and immunohistochemistry assays of IL-6. Representative images were captured. Scale bar, 200 μm. N = 5–6 mice/group. * p < .05, ** p < .01, *** p < .001, compared with HFC control mice
Figure 1. A. muciniphila attenuated obese MAFLD induced by HFC diet feeding. (a-h) Eight-week-old male C57BL/6 mice were fed with an HFC diet for 11 weeks to induce obese MAFLD. They were then randomly divided into two subgroups to treat with PBS as a control (HFC group) or A. muciniphila in PBS (HFC+A. muciniphila) orally every other day for 6 weeks. (a) Schematic diagram of A. muciniphila treatment. (b) Abundance of A. muciniphila in feces. (c) Mouse body weight at week 17. (d-e) Hepatic triglyceride (TG) and diacylglyceride (DAG) levels. (f) Plasma levels of AST, ALT and ALP. (g) Pathologic examination of the liver by hematoxylin ＆ eosin (H & E) staining, oil red O staining (ORO), and immunofluorescence examination of IL-6 and CD68. Representative images were captured. Scale bar, 200 μm. (h) Expression of mRNA markers for steatosis and inflammation markers. The levels of genes in the HFC control group were set as 1, and the relative fold increases were determined by comparison with the HFC control group. N = 5–8 mice/group. * p < .05, compared with HFC control group mice. (i-m) Sustainability of the anti-MAFLD efficacy of A. muciniphila after the cessation of its administration. (i) Schematic diagram of the experimental design. After 6 weeks of administration, A. muciniphila was withdrawn from the HFC-induced obese MAFLD mice followed by a period of 4 weeks of washout to evaluate of the sustainability of the anti-MAFLD efficacy. (j) Mouse body weight. (k) TG level in the liver. (l) Plasma levels of AST, ALT and ALP. (m) Pathologic examination of the liver by H ＆ E staining, oil red O staining, and immunohistochemistry assays of IL-6. Representative images were captured. Scale bar, 200 μm. N = 5–6 mice/group. * p < .05, ** p < .01, *** p < .001, compared with HFC control miceCC BY · PubMed Central
Figure 2. A. muciniphila increased markers related to lipid metabolism in liver and gut of HFC mice. HFC induced obese MAFLD (11 weeks of feeding) were administered with saline as the control or A. muciniphila (6 weeks of treatment) to assess liver and ileum parameters: a-c for parameters in the liver and d-f for parameters in the ileum. e-g, representative images of the ileum (Scale bar, for 100 µm). (a) Hepatic mitochondrial copy number determination. (b) Gene expression related to lipid uptake and oxidation in the liver of mice. (c) Protein levels of metabolic regulators mitochondrial complexes, and the LKB1-AMPK axis in the liver of mice. The protein levels were quantified and normalized to the loading control actin (d) Gene expression of lipid uptake and oxidation in the ileum of mice. The gene level or protein level in the HFC group was set as 1, and the relative fold increases were determined by comparison with the HFC control group. (e) Immunohistochemistry analysis of PGC-1α in the ileum of mice. The brown dot indicates the examined protein. Representative images were captured. Scale bar, 100 µm. (f) TG level quantification (indicated by oil red O staining) and oxidative stress-induced cell apoptosis determination (indicated by CHOP examination) in the ileum. Representative images were captured. Scale bar, 100 µm. (g) Immunohistochemistry analysis of E-cadherin and H＆E examination in ileum of HFC mice. The brown dot indicates the target protein. Representative images were captured. Scale bar, 100 µm. N = 5–8 mice/group. * p < .05, ** p < .01, *** p < .001, compared with HFC control mice
Figure 2. A. muciniphila increased markers related to lipid metabolism in liver and gut of HFC mice. HFC induced obese MAFLD (11 weeks of feeding) were administered with saline as the control or A. muciniphila (6 weeks of treatment) to assess liver and ileum parameters: a-c for parameters in the liver and d-f for parameters in the ileum. e-g, representative images of the ileum (Scale bar, for 100 µm). (a) Hepatic mitochondrial copy number determination. (b) Gene expression related to lipid uptake and oxidation in the liver of mice. (c) Protein levels of metabolic regulators mitochondrial complexes, and the LKB1-AMPK axis in the liver of mice. The protein levels were quantified and normalized to the loading control actin (d) Gene expression of lipid uptake and oxidation in the ileum of mice. The gene level or protein level in the HFC group was set as 1, and the relative fold increases were determined by comparison with the HFC control group. (e) Immunohistochemistry analysis of PGC-1α in the ileum of mice. The brown dot indicates the examined protein. Representative images were captured. Scale bar, 100 µm. (f) TG level quantification (indicated by oil red O staining) and oxidative stress-induced cell apoptosis determination (indicated by CHOP examination) in the ileum. Representative images were captured. Scale bar, 100 µm. (g) Immunohistochemistry analysis of E-cadherin and H＆E examination in ileum of HFC mice. The brown dot indicates the target protein. Representative images were captured. Scale bar, 100 µm. N = 5–8 mice/group. * p < .05, ** p < .01, *** p < .001, compared with HFC control miceCC BY · PubMed Central
Figure 3. A. muciniphila promoted the metabolism of bile acid in the liver and gut of HFC mice. HFC-induced obese MAFLD (11 weeks of feeding) were administered saline as the control or A. muciniphila (6 weeks of treatment) to assess the liver and ileum parameters. (a) Schematic diagram of bile acid shuttling between the liver and gut. (b) Expression of mRNAs involved in the regulation of the synthesis and transport of bile acids in the liver. (c) Expression of mRNAs involved in the regulation of the synthesis and transport of bile acids in the ileum (d) Plasma level of cholesterol in plasma or the liver. (e) mRNA levels of mucoprotein-related genes in the colon of HFC mice. The level of genes in the HFC control group were set as 1, and the relative fold increases were determined by comparison with the HFC control group. N = 5–8 mice/group. * p < .05, ** p < .01, *** p < .001, compared with the HFC control group mice
Figure 3. A. muciniphila promoted the metabolism of bile acid in the liver and gut of HFC mice. HFC-induced obese MAFLD (11 weeks of feeding) were administered saline as the control or A. muciniphila (6 weeks of treatment) to assess the liver and ileum parameters. (a) Schematic diagram of bile acid shuttling between the liver and gut. (b) Expression of mRNAs involved in the regulation of the synthesis and transport of bile acids in the liver. (c) Expression of mRNAs involved in the regulation of the synthesis and transport of bile acids in the ileum (d) Plasma level of cholesterol in plasma or the liver. (e) mRNA levels of mucoprotein-related genes in the colon of HFC mice. The level of genes in the HFC control group were set as 1, and the relative fold increases were determined by comparison with the HFC control group. N = 5–8 mice/group. * p < .05, ** p < .01, *** p < .001, compared with the HFC control group miceCC BY · PubMed Central
Figure 4. A. muciniphila increased L-aspartate level in liver of HFC mice. HFC-induced obese MAFLD (11 weeks of feeding) were administered saline (HFC), A. muciniphila, antibiotics (Abx) or a combination of Abx plus A. muciniphila (Abx+A. muciniphila) for (6 weeks of treatment) to assess liver metabolomics and the indicated assays. (a) PCA analysis. (b) Identification of significant differentially metabolites in A. muciniphila- or Abx+A. muciniphila-treated HFC mice compared with HFC or Abx-treated HFC mice by LC-MS analysis. Significant differentially metabolites were identified based on the criteria of fold ≥ 2 and a Q value equal to or higher than 0.05. The dots represent differentially metabolites. N = 5 mice/group. (c) Annotation of the identified significant metabolites in the HFC, HFC+A. muciniphila, HFC+Abx, and HFC+Abx+A. muciniphila groups by metabolomics trend analysis. (d) Comparative analysis of the hepatic L-aspartate and TG levels. (e) L-aspartate level in the feces and plasma of mice. N = 5 mice/group. (f) Gene expression of Slc1a1 in the ileum of mice. (g) Gene expression of Slc1a2 in the liver of mice. The level of mRNA in the HFC control group was set as 1, and relative fold increases were determined by comparison with the HFC control group. N = 5–8 mice/group. * p < .05, ** p < .01, *** p < .001, compared with HFC control mice. # p < .05, ## p < .05, ### p < .001, compared with antibiotics (Abx)-treated HFC mice; $ p < .05, $$ p < .01, $$$ p < .001, compared with A. muciniphila-treated HFC mice
Figure 4. A. muciniphila increased L-aspartate level in liver of HFC mice. HFC-induced obese MAFLD (11 weeks of feeding) were administered saline (HFC), A. muciniphila, antibiotics (Abx) or a combination of Abx plus A. muciniphila (Abx+A. muciniphila) for (6 weeks of treatment) to assess liver metabolomics and the indicated assays. (a) PCA analysis. (b) Identification of significant differentially metabolites in A. muciniphila- or Abx+A. muciniphila-treated HFC mice compared with HFC or Abx-treated HFC mice by LC-MS analysis. Significant differentially metabolites were identified based on the criteria of fold ≥ 2 and a Q value equal to or higher than 0.05. The dots represent differentially metabolites. N = 5 mice/group. (c) Annotation of the identified significant metabolites in the HFC, HFC+A. muciniphila, HFC+Abx, and HFC+Abx+A. muciniphila groups by metabolomics trend analysis. (d) Comparative analysis of the hepatic L-aspartate and TG levels. (e) L-aspartate level in the feces and plasma of mice. N = 5 mice/group. (f) Gene expression of Slc1a1 in the ileum of mice. (g) Gene expression of Slc1a2 in the liver of mice. The level of mRNA in the HFC control group was set as 1, and relative fold increases were determined by comparison with the HFC control group. N = 5–8 mice/group. * p < .05, ** p < .01, *** p < .001, compared with HFC control mice. # p < .05, ## p < .05, ### p < .001, compared with antibiotics (Abx)-treated HFC mice; $ p < .05, $$ p < .01, $$$ p < .001, compared with A. muciniphila-treated HFC miceCC BY · PubMed Central
Figure 5. L-aspartate reduced lipid and ROS in hepatocytes along with increases in LKB1-AMPK activity and mitochondrial oxidative capacity. L-02 cells induced with oleatic acid (0.75 mM) were treated with L-aspartate (10, 40 µM) for 24 h, and then cells were harvested for the indicated analysis. (a) TG level in cell lysates. (b) Nile red staining using a Nile red probe at a final concentration of 5 μM. (c) Key proteins in the LKB1-AMPK axis and mitochondrial oxidation. The protein levels were quantified against the loading control GAPDH. The protein levels in control cells of each independent experiment were set as 1, and the relative fold increases were determined by comparison with the control group. (d-e) Quantification of the ROS levels by flowcytometry or confocal microscopy using the DCFH-DA probe at a final concentration of 10 μM. Representative images were captured. Scale bar, 100 µm. The data shown are individual values with means ± SEM. N = 5 independent experiments. * p < .05, ** p < .01, *** p < .001, compared with control group cells
Figure 5. L-aspartate reduced lipid and ROS in hepatocytes along with increases in LKB1-AMPK activity and mitochondrial oxidative capacity. L-02 cells induced with oleatic acid (0.75 mM) were treated with L-aspartate (10, 40 µM) for 24 h, and then cells were harvested for the indicated analysis. (a) TG level in cell lysates. (b) Nile red staining using a Nile red probe at a final concentration of 5 μM. (c) Key proteins in the LKB1-AMPK axis and mitochondrial oxidation. The protein levels were quantified against the loading control GAPDH. The protein levels in control cells of each independent experiment were set as 1, and the relative fold increases were determined by comparison with the control group. (d-e) Quantification of the ROS levels by flowcytometry or confocal microscopy using the DCFH-DA probe at a final concentration of 10 μM. Representative images were captured. Scale bar, 100 µm. The data shown are individual values with means ± SEM. N = 5 independent experiments. * p < .05, ** p < .01, *** p < .001, compared with control group cellsCC BY · PubMed Central
Figure 6. Proposed mechanism by which A. muciniphila ameliorates MAFLD in HFC diet-induced obese mice. A. muciniphila treatment increases L-aspartate levels in the gut-liver axis, which activates the LKB1-AMPK axis and increases lipid oxidation and bile acid metabolism in the gut-liver axis, leading to decreased lipid accumulation and oxidation stress-induced cell apoptosis. These metabolic improvements result in maintaining the integrity of the gut barrier and reshaping the gut microbiota composition for MAFLD treatment in mice
Figure 6. Proposed mechanism by which A. muciniphila ameliorates MAFLD in HFC diet-induced obese mice. A. muciniphila treatment increases L-aspartate levels in the gut-liver axis, which activates the LKB1-AMPK axis and increases lipid oxidation and bile acid metabolism in the gut-liver axis, leading to decreased lipid accumulation and oxidation stress-induced cell apoptosis. These metabolic improvements result in maintaining the integrity of the gut barrier and reshaping the gut microbiota composition for MAFLD treatment in miceCC BY · PubMed Central

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Metadata

Authors
Rao, Y., Kuang, Z., Li, C., Guo, S., Xu, Y., Zhao, D., Hu, Y., Song, B., Jiang, Z., Ge, Z., Liu, X., Li, C., Chen, S., Ye, J., Huang, Z., & Lu, Y.
Affiliations
  • Sun Yat-sen University (CN)
  • Sun Yat-sen University
  • RMIT University
Corresponding author
  • Rao, Yong
  • Lu, Yongjun
ORCID
Venue
Gut Microbes
Publisher
Informa UK Limited
Published
2021-01-01
Volume
13
Issue
1
Pages
1-19
ISSN
  • 1949-0976 (ISSN-L)
  • 1949-0984
DOI
10.1080/19490976.2021.1927633
PMC
PMC8158032
OpenAlex
W3165210923
Cited by
356
References
70
Citations by year
2026: 65 · 2025: 112 · 2024: 67 · 2023: 51 · 2022: 46 · 2021: 14
FWCI
13.97
Type
journal article
Language
en
Open access
Licence
Locations
  • Gut Microbes (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RMIT Research Repository (RMIT University Library) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion)
Funders
  • National Natural Science Foundation of China (81930098 to ZSH, 21672265 and 81872732 to ZSH, 81703336 to YR)
  • Science and Technology Program of Guangzhou (201704020104 to ZSH)
  • Science and Technology Development in Guangdong Province (2016A020217004 to ZSH)
  • Natural Science Foundation of Guangdong Province (2017A030308003 to ZSH)
  • Guang Dong Cheung Kong Philanthropy Foundation to YJL
  • Fundamental Research Funds for the Central Universities
  • Guang Dong Cheung Kong Philanthropy Foundation
  • Outstanding Talents of Guangdong Special Plan (2019JC05Y456)
  • the Science and Technology Program of Guangzhou (201704020104)
  • Special Fund for Science and Technology Development in Guangdong Province (2016A020217004)
  • the Natural Science Foundation of Guangdong Province (2017A030308003)
  • Guangdong key areas R & D projects (2018B020205002)
  • Key projects of National Natural Science Foundation of China (81930098)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Health Sciences › Medicine › Hepatology › Liver Disease Diagnosis and Treatment
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Diet, Metabolism, and Disease
Keywords
Akkermansia muciniphila, non-alcoholic fatty liver disease, L-aspartic acid, gut-liver axis, bile acid metabolism, hepatic steatosis, oxidative stress, apoptosis, gut microbiota, high-fat high-cholesterol diet, obesity, mitochondrial oxidation, antibiotic-treated mice
MeSH terms
Gastrointestinal Microbiome, Akkermansia, Animals, Aspartic Acid, Bacteria, Fatty Liver, Humans, Liver, Male, Mice, Inbred C57BL, Gastrointestinal Tract, Mice, Diet, High-Fat

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