Modulating phenylalanine metabolism by L. acidophilus alleviates alcohol-related liver disease through enhancing intestinal barrier function
Cell & Bioscience volume 13, Article number: 24 (2023)
Impaired metabolic functions of gut microbiota have been demonstrated in alcohol-related liver disease (ALD), but little is known about changes in phenylalanine metabolism.
Bacterial genomics and fecal metabolomics analysis were used to recognize the changes of phenylalanine metabolism and its relationship with intestinal flora. Intestinal barrier function was detected by intestinal alkaline phosphatase (IAP) activity, levels of tight junction protein expression, colonic inflammation and levels of serum LPS. Lactobacillus acidophilus was chosen to correct phenylalanine metabolism of ALD mice by redundancy analysis and Pearson correlation analysis.
Using 16S rRNA sequencing and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods, we identified elevated levels of phenylalanine and its’ metabolites in the gut of alcohol-fed mice compared to control mice and were negatively correlated with the abundance of Lactobacillus, which mainly metabolized phenylalanine. The intestinal phenylalanine level was positively correlated with the colon inflammatory factors TNF-α and IL-6, and negatively correlated with ZO-1 and Occludin. While intestinal alkaline phosphatase (IAP) activity was negatively correlated with the colon inflammatory factors TNF-α, IL-6 and MCP-1, and positively correlated with ZO-1 and Occludin. Increased phenylalanine inhibited IAP activity, blocked LPS dephosphorylation, increased colonic inflammation and bacterial translocation. Phenylalanine supplementation aggravated alcohol-induced liver injury and intestinal barrier dysfunction. Among the 37 Lactobacillus species, the abundance of Lactobacillus acidophilus was most significantly decreased in ALD mice. Supplementation with L. acidophilus recovered phenylalanine metabolism and protected mice from alcohol-induced steatohepatitis.
Recovery of phenylalanine metabolism through the oral supplementation of L. acidophilus boosted intestinal barrier integrity and ameliorated experimental ALD.
Gut dysbiosis has been shown to participate in the progression of alcohol-related liver diseases (ALD). Germ-free mice exposed to drinking water supplemented with alcohol for 7 days did not have liver injury or hepatic inflammation compared with conventional mice . Germ-free mice transplanted with the intestinal microbiota of patients with severe alcoholic hepatitis had a more severe liver injury and inflammation and increased intestinal permeability than those transplanted with the intestinal microbiota of patients with no alcoholic hepatitis . Alcohol administration reduced bacterial diversity and uniquely shaped the composition of the intestinal bacteria. Alcohol-dependent individuals showed a decrease in the species of Bifidobacterium and an increase in the genera of Dorea, Blautia and Megasphaera compared with controls . Patients with alcoholic hepatitis showed a significant increase in the abundance of Enterococcus species in feces, compared with controls . Consistent with previous studies [5,6,7], we also found alcohol-treated mice had reduced abundances of the Lactobacillus and Akkermansia genera, compared with pair-fed mice.
Alcohol-induced functional changes in the intestinal microbiota are also important for the pathogenesis of alcoholic liver disease. Short-chain fatty acids (SCFAs), which are by-products of fermented fiber by intestinal bacteria, showed a significant decrease in the colon of rats chronically exposed to alcohol, compared with control rats . Supplementation of tributyrin and propionate improved the intestinal barrier function and alleviated liver injury in the chronic-binge alcohol-fed mice [9, 10]. Chronic administration of ethanol inhibited bacterial gene expression involved in the biosynthesis of saturated fatty acids, compared to controls, and dietary supplementation of saturated long chain fatty acids prevented alcohol-induced gut leakiness and liver damage . Chronic-binge alcohol treatment inhibited bacterial catabolism of tryptophan into indoles, reduced REG3G expression in enterocytes and increased intestinal bacterial translocation . Serum concentrations of tryptophan metabolites decreased in patients with severe alcoholic hepatitis compared to alcoholic patients with hepatitis . Although the role of altered aromatic amino acid metabolism has been demonstrated in alcohol-induced liver damage, little is known about changes in phenylalanine metabolism.
In our previous study, we found that intestinal L-phenylalanine levels increased significantly in alcohol-fed mice, compared to control mice. Similar findings have reported that chronic alcohol consumption increases the level of L- phenylalanine in the feces of mice . Liver and serum levels of phenylalanine were found to be elevated in patients with alcohol-associated liver damage [14, 15], but decreased in the feces of patients with actively drinking cirrhosis . L-phenylalanine has been proven to act as a selective inhibitor of epithelial alkaline phosphatase . Intestinal alkaline phosphatase (IAP) has shown many physiological functions, including reduced endotoxemia and subsequent inflammation, and maintenance of intestinal barrier function . Aging mice that showed a decreased in IAP activity had high intestinal permeability and systemic inflammation . Oral IAP supplementation improved intestinal barrier function, attenuated alcohol-induced liver steatosis and inflammation , and rescued bile duct ligation, and carbon tetrachloride-4 (CCl4)-induced liver fibrosis . In addition to the inhibitory effect of phenylalanine on IAP, its metabolites produced by intestinal bacteria also act as modulators of gastrointestinal function. In this study, we examined the role of alcohol-induced abnormal phenylalanine metabolism in intestinal barrier injury and liver injury. We also investigated how Lactobacillus acidophilus supplementation restored phenylalanine metabolism and offered protection from ethanol-induced hepatic steatosis.
Eight weeks-old male C57BL/6 mice were purchased from Vital River Labs (Beijing, China). A Lieber-De Carli diet was used to build an acute-on chronic alcohol exposure model based on the results of a previous study . Briefly, the mice were first acclimated to a liquid diet for 5 days, then fed on a Lieber-De Carli diet containing 5 vol% ethanol for 10 days. Finally, they were binged with 5 g of alcohol per kilogram of body weight. The control group of mice was fed maltose dextrin containing the same calories instead of ethanol. Tissues and serum were collected 9 h after the binge for follow-up experiments.
For phenylalanine supplementation, mice fed ethanol and isocaloric were gavaged daily with 200 mg/Kg phenylalanine or PBS for a total of 15 days.
For Lactobacillus treatment, mice fed ethanol and isocaloric were gavaged daily with L. acidophilus (5 × 108 colony-forming units (CFU) in 200 μl phosphate-buffered saline (PBS)) or PBS for a total of 15 days. L. acidophilus (ATCC 43,550) was purchased from the Shanghai Conservation Biotechnology Center and incubated in the MRS broth medium at 37 ℃.
The animal studies complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
16S rRNA sequencing
The caecal stool was collected and DNA was extracted from the ethanol-fed and isocaloric-fed mice with and without L. acidophilus treatment. The V3-V4 hypervariable regions of 16S rRNA were amplified. Sequencing libraries were generated using NEB Next® Ultra DNA Library Prep Kit (Illumina, USA). At last, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads were generated. QIIME software was used to construct the operational taxonomic unit (OUT) abundance tables of each sample. The alpha diversity of the intestinal microbiota was measured using the Shannon and Chao indexes, and the beta diversity of the intestinal microbiota was analyzed by principal coordinate analysis (PCoA) and the partial least squares discriminant analysis (PLS-DA). The linear discriminant analysis effect size (LEfSe) was applied to identify species with significant differences in abundance between the groups using the Wilcoxon rank sum test. Comparison with sequencing data on known metagenomic functional data was performed using PICRUSt software, while KEGG metabolic pathways were used to predict differences in functional genes of microbial communities between the groups. To identify bacteria species associated with phenylalanine metabolism, redundancy analysis (RDA) were performed.
Untargeted metabolome analysis
Fecal samples were obtained from the ethanol-fed and isocaloric-fed mice with and without L. acidophilus treatment and analyzed using UPLC-MS/MS at positive ion mode electrospray ionization (ESI) and negative ion mode ESI. Raw data were processed for quality control and normalization, and then the PLS-DA model was used to discriminate significant differences between the groups. The differences in metabolite levels between the groups were compared using the Student's t test.
Measurement of intestinal alkaline phosphatase (IAP) and LPS dephosphorylation activities
The modified Gomori calcium-cobalt method was used to measure IAP activity. According to the instructions provided with the kit (Solarbio, Beijing, China), paraffin-embedded colon tissues were sliced and dewaxed. The samples were then incubated with sodium glycerol phosphate solution or LPS solution (1 mg/ml) for 4 h, and cobalt buffer for 5 min at 37 ℃. Hydrogen sulfide working buffer was added to the samples and allowed to react for 2 min, and then rinsed with distilled water. Finally, nuclear fast red was used to visualize the nuclei.
Liver tissues were stained with hematoxylin eosin (HE) to observe signs of liver injury. To assess liver steatosis, liver tissues were embedded in OCT, and cut to a thickness of 10 μm. The frozen sections were then stained with oil red O (Sigma-Aldrich, Shanghai, China).
Serum ALT, AST, and LPS detection
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Endotoxin levels were examined using a LPS ELISA kit (Solarbio, Beijing, China) according to the protocols suggested by the manufacturer.
Immunohistochemistry (IHC) and immunofluorescence (IF)
Immunohistochemical staining was performed using a goat anti-rabbit IgG (H + L) IHC kit (Servicebio, Wuhan, China). Colon slices were incubated with F4/80 (1:300, Servicebio), then stained with a DAB working solution, while the nucleoli were stained with hematoxylin. For IF staining, colon tissues were incubated with LPS (1:50, Hycult Biotech, Netherlands), ZO-1 (1:200, Servicebio, Wuhan, China), and Occludin (1:200, Proteintech, Chicago, United States) antibodies. The slices were then stained with the Alexa Fluor 594 conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:500, CST, Massachusetts, USA), and Alexa Fluor 594 conjugated goat anti-Mouse IgG (H + L) secondary antibody (1:500, CST). The nuclei were stained with DAPI.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from colon tissues of ethanol-fed and control mice with and without L. acidophilus supplementation using TRIzol reagent (Invitrogen, California, USA). Total RNA was then reverse transscribed using the PrimeScript™ RT Master Mix (RR036A, TaKaRa). PCR amplifications were performed on a LightCycler 96 amplifier using the PowerUp™ SYBR™ Green Master Mix (Invitrogen, Carlsbad, CA). The detection of liver translocation bacteria was carried out using previously published methods [11, 23]. The primer sequences used are shown in Additional file 1: Table S1.
Cell culture and treatment
Primary hepatocytes were isolated from C57BL/6 mice according to previous study . Starved hepatocytes (3% fetal bovine serum) were stimulated with ethanol (50 mM) and phenylalanine (100 μM) for 24 h. Hepatocyte cytotoxicity was measured using the lactate dehydrogenase (LDH) cytotoxicity assay kit (Beyotime Biotechnology, Shanghai, China).
Human colon epithelial cells (NCM460) were cultured in DMEM medium with 10% fetal bovine serum. Then NCM460 cells were starved with 3% fetal bovine serum and treated with ethanol (50 mM) and phenylalanine (100 μM) for 24 h. The IAP expression was measured by immunofluorescence (1:300, Servicebio, Wuhan, China), and ZO-1 and Occludin expressions were measured by western blotting.
Fecal lactobacillus species identification
A total of 37 lactobacillus species were identified and compared in fecal samples obtained from ethanol-fed and isocaloric-fed mice, based on a previous study . Total fecal DNA was extracted using a fecal genomic DNA extraction kit (TIANGEN, Beijing, China). Species-specific primer sequences and PCR amplification conditions used in previous study were used .
In vivo intestinal permeability assay
Intestinal permeability was assessed by gastric gavage of fluorescein isothiocyanate-dextran (FITC-dextran) (4 kDa; Sigma-Aldrich). Mice were gavaged with 200 μL of FITC-dextran (100 mg/mL) 5 h after ethanol binge, and the blood samples were collected 4 h later. Serum FITC-dextran levels were measured using a fluorimeter.
Data were shown as mean ± SEM. Student’s t test and Mann-Whitney test were used to analyze the differences between groups. P value of less than 0.05 was considered to indicate significance.
Phenylalanine metabolism by intestinal bacteria was disordered in the ethanol-fed mice
The alpha diversities of intestinal bacteria determined using the Shannon and Chao indices were not significantly different between the chronic–binge ethanol-fed mice and the control mice (Fig. 1A). However, the principal coordinate analysis (PCoA) and the partial least squares discriminant analysis (PLS-DA) revealed that the bacterial composition was significantly different between the mice fed ethanol and the control mice (Fig. 1B, C). Consistent with the results of previous studies , the abundance of Akkermansia muciniphila decreased significantly in the chronic–binge ethanol-fed mice (Fig. 1D). We also observed that the Lactobacillus genus showed a prominent decline.
To explore the functions of intestinal bacteria, we performed a KEGG pathway analysis. Among the changes in amino acid metabolism, which were the focus of this study, a decrease in phenylalanine metabolism of ethanol feeding mice caught our attention (Fig. 2A). This result was supported by our previous findings on elevated phenylalanine levels in the gut of alcohol-fed mice, compared to control mice (Fig. 2B). Two metabolites were detected in the fecal matter of the ethanol-fed and control mice. The 3-phenyllactic acid level increased in the ethanol-fed mice in contrast to the control mice, but the phenylpyruvate level did not show significant differences (Fig. 2B). The ratios of phenylpyruvate to L-phenylalanine and 3-phenyllactic acid to L-phenylalanine were lower in the alcohol-fed mice than in the control mice (Fig. 2C). The abundance of bacterial D-lactate dehydrogenase (D-LDH), which has been reported to metabolize phenylpyruvic acid into phenyllactic acid [26,27,28], was lower in the alcohol-fed mice than in the control mice (Fig. 2D, E). The level of bacterial phenylacetaldehyde dehydrogenase was higher in alcohol-fed mice than in control mice.
High levels of phenylalanine inhibited intestinal alkaline phosphatase activity (IAP) and correlated with the colonic inflammatory injury of ethanol feeding mice
IAP expressed on the apical surfaces of enterocytes plays an important role in detoxifying LPS and protecting the intestinal barrier . L-phenylalanine is known as an inhibitor of IAP . Then, we measured the activity of IAP in the chronic–binge ethanol-fed mice and control mice. As shown in Fig. 3A, colonic IAP activity had decreased significantly in chronic–binge ethanol-fed mice compared to control mice. The ability of IAP to dephosphorylate and inactivate LPS also decreased in the alcohol-fed mice (Fig. 3B). Consistently, colonic bacterial invasion detected by immunofluorescence with anti-LSP antibody increased in mice fed alcohol, compared to control mice (Fig. 3C). Furthermore, an increase in pro-inflammatory macrophage infiltration into the colon was observed in alcohol-fed mice, compared to controls (Fig. 3D, Additional file 1: Figure S1).
To determine whether the increase in colonic inflammatory reaction was related to the high level of phenylalanine and decreased IAP activity, we analyzed the correlation of intestinal phenylalanine content and IAP activity, with the expression levels of inflammatory factors and tight junction proteins. The level of phenylalanine was positively correlated with the mRNA levels of TNF-α, and IL-6, and negatively correlated with the mRNA levels of ZO-1 and Occludin (Tables 1 and 2). While IAP activity was negatively correlated with the mRNA levels of TNF-α, IL-1, MCP-1, and positively correlated with the mRNA levels of ZO-1 and Occludin (Tables 1 and 2).
Phenylalanine supplementation aggravated alcohol-induced liver injury and intestinal barrier dysfunction
To further evaluate whether phenylalanine was responsible for alcohol-related intestinal barrier function impairment, alcohol-fed and isocaloric-fed mice were treated with phenylalanine. As shown in Fig. 4A–C, mice with phenylalanine supplementation had more severe alcohol-induced liver injury and steatosis. Intestinal permeability measured by level of serum LPS was increased in alcohol-exposed mice supplemented with phenylalanine, compared to mice supplemented with PBS (Fig. 4D). In addition, phenylalanine is associated with increased colonic inflammation. TNF-α, MCP-1, IL-6 and IL-1 expressions, and macrophage infiltration were increased in alcohol plus phenylalanine treated mice, as compared to alcohol plus PBS treated mice (Fig. 4E, F).
In vitro, primary mouse hepatocytes were isolated and treated with alcohol or phenylalanine. Phenylalanine did not affect hepatocytes cytotoxicity caused by alcohol (Fig. 4G). Phenylalanine intervention exacerbated reduction of alcohol-induced IAP expression of human colon epithelial cells (NCM460) (Fig. 4H). The expressions of tight junction proteins ZO-1 and Occludin were also decreased in ethanol plus phenylalanine treated NCM460 cells compared to ethanol plus PBS treated NCM460 cells (Fig. 4I).
Decreased abundance of Lactobacillus related with abnormal metabolism of phenylalanine
Redundancy analysis (RDA) using bacteria species that different between alcohol-fed and control mice, and phenylalanine and its’ metabolites, showed only Lactobacillus was obviously negative related with phenylalanine and its metabolites phenylpyruvate and 3-phenyllactic acid (Fig. 5A). Although alcohol exposure can cause a greater depletion of Akkermansia than Lactobacillus, only Lactobacillus was positively correlated with intestinal phenylalanine activity (Fig. 5B). To further identify the Lactobacillus species, which was affected by alcohol feed, 37 species-specific primer pairs were selected and used to differentiate species using PCR assays based on methods previously described . Seven Lactobacillus species were detected in at least four samples from mice fed ethanol (Additional file 1: Table S2, Fig. 5C). The alcohol-fed mice showed a decrease in L. reuteri, L. johnsonii, and L. acidophilus, compared with the control mice, especially L. acidophilus, since it was not detected in alcohol-fed mice (CT value > 40). L. fermentum abundance was higher in alcohol-fed mice, compared to control mice. On the contrary, there were no significant differences in L. paracasei, L. delbrueckii, and L. brevis abundances between the alcohol-fed mice and the control mice.
Supplementation with L. acidophilus alleviated ethanol-induced liver injury and intestinal barrier damage
Since L. acidophilus has been shown to express D-LDH  and is one of the main bacteria metabolizing phenylalanine , we hypothesized that L. acidophilus administration may ameliorate alcohol-induced liver injury. Oral L. acidophilus supplementation attenuated liver steatosis and liver damage, as demonstrated by the significant reduction in serum levels of ALT and AST and the accumulation of liver lipid droplets (Fig. 6A–C). IF staining of ZO-1 and Occludin revealed that chronic-binge ethanol feeding injured the colonic barrier of the mice, and L. acidophilus supplementation reduced damage of the intestinal barrier function (Fig. 6D, E). Consistent with the expression of tight junction proteins, an increase in serum levels of LPS, FITC labeled dextran (4KD), and liver bacteria was found in alcohol-exposed mice compared to control mice, and decreased after L. acidophilus supplementation (Fig. 6F–H).
Supplementation with L. acidophilus ameliorated ethanol-induced inflammatory injury of colon
To further elucidate the effect of L. acidophilus on the colonic inflammatory response, colonic IAP activity and LPS dephosphorylation levels were measured. Ethanol consumption decreased IAP activity and the level of LPS dephosphorylation, while L. acidophilus supplementation could recover IAP activity and LPS dephosphorylation (Fig. 7A, B). Furthermore, bacterial invasion decreased when chronic-binge ethanol-fed mice were supplemented with L. acidophilus (Fig. 7C). Given that IAP can prevent inflammation, macrophages infiltration levels and inflammatory factors were measured. Alcohol exposure caused inflammation in the colon, characterized by macrophage infiltration and increased expression of inflammatory factors, compared to controls (Fig. 7D, E). L. acidophilus supplementation decreased colonic inflammation by reducing macrophage infiltration levels and the expression levels of the inflammatory factors TNF-α, TGF-β, MCP-1, and IL-6 (Fig. 7D, E).
L. acidophilus supplementation restored the dysfunction of alcohol- associated phenylalanine metabolism
16S rDNA sequencing of the stool was performed to determine the effect of L. acidophilus supplementation on the intestinal microbiota in alcohol-fed mice. As shown in Fig. 8A, the bacterial diversity calculated using the Shannon and Chao indexes was similar in ethanol feeding mice with or without L. acidophilus supplementation. The PLS-DA analysis showed that mice fed ethanol with or without L. acidophilus supplementation were clustered differently (Fig. 8B). Some bacteria classifications were significantly changed in the ethanol-fed mice with L. acidophilus supplementation, compared to mice without L. acidophilus supplementation (Fig. 8C, Additional file 1: Figure S2.). Typically, the abundance of the Lactobacillus genus (Lactobacillaceae family) increased, while the abundance of the Parabacteroides genus (Porphyromonadaceae family) decreased after L. acidophilus supplementation. Phenylalanine metabolism and the abundances of bacterial D-lactate dehydrogenase and phenylacetaldehyde dehydrogenase were increased significantly in mice supplemented with L. acidophilus, compared to mice not supplemented with L. acidophilus (Fig. 8D, E).
The fecal metabolites of mice fed ethanol and supplemented with L. acidophilus were different from those of mice without L. acidophilus supplementation (Fig. 9A). The intestinal phenylalanine level decreased after the alcohol-fed mice were treated with L. acidophilus (Fig. 9B), but did not show any differences in the level of phenylpyruvate and 3-phenyllactic acid. And the ratios of phenylpyruvate to L-phenylalanine and 3-phenyllactic acid to L-phenylalanine were same between alcohol-fed mice with and without L. acidophilus supplementation (Fig. 9C).
Phenylalanine, an essential amino acid, is converted to tyrosine by phenylalanine hydroxylase in the liver. Subsequently, it is further metabolized into neurotransmitters, such as dopamine (DA), norepinephrine (NE), adrenaline (E), and melanin, which regulate the metabolism of glucose and fat in the human body . In vitro, phenylalanine inhibits hepatocyte glycolysis but does not affect mitochondrial respiration by inhibiting pyruvate kinase and hexokinase, which are key enzymes of the glycolytic pathway . There was a significant increase in intestinal phenylalanine level in patients with colorectal intramucosal carcinomas, compared with healthy controls . However, in ALD, changes in intestinal phenylalanine and its metabolism by bacteria have been not reported. We found that mice exposed to alcohol had a higher level of intestinal phenylalanine and a lower abundance of bacterial enzymes that metabolize phenylalanine, compared to the control mice.
IAP has been found mediating interactions between microorganism communities and the host. IAP dephosphorylates LPS and other pathogen-associated molecular patterns, reducing local intestinal inflammation  and helping maintain gut barrier function [29, 36]. IAP supplementation has been reported to be able to ameliorate experimental colitis , reverse metabolic syndrome , prevent liver fibrosis , and attenuate alcohol-induced hepatosteatosis . Decreased IAP expression is associated with impaired barrier function. We found supplement with phenylalanine, a known inhibitor of IAP, exacerbated intestinal barrier function damage of chronic–binge ethanol-fed mice.
Bacterial metabolites of phenylalanine also regulate intestinal and liver homeostasis. Phenyllactic acid (PLA) is an attractive organic acid with a broad antibacterial and antifungal spectrum . A previous study found that the excretion rate of phenyllactic acid in the urine of C57BL/6N mice exposed to alcohol for 3 months had increased significantly, while that of indole-3-lactic acid remained unchanged . Hepatocellular carcinoma patients had significantly higher levels of 3-phenyllactic acid in the portal vein and cancerous tissues than healthy controls, which can be associated with impaired liver function and poor survival . Phenylacetic acid (PAA) triggered hepatic steatosis and increased branched-chain amino acid utilization . In healthy individuals, fecal PAA was found to be negatively associated with the Clostridium XIVa cluster and positively with Lactobacillus . Individuals with high levels of fecal phenylpropionate had elevated serum levels of TGF-β, IL-17, IL-8, malondialdehyde and C-reactive protein, and were in a low pro-oxidation and pro-inflammatory state . However, changes in these metabolites in the circulatory system and liver of alcohol-fed mice and control mice were not measured in our study. The direct role of phenylalanine metabolites in alcohol-induced liver injury deserves further exploration.
Probiotic Lactobacilli has been found to ameliorate ethanol-induced hepatic damage via several ways . Lactobacillus rhamnosus GG treatment recovered intestinal barrier functions of ALD mice through increasing tight junction protein expressions [44, 45]. Lactobacillus rhamnosus GG culture supernatant restored alcohol-induced intestinal barrier dysfunction by promoting HIF signaling . Long time Lactobacillus rhamnosus GG supplementation prevented alcohol-induced colonic dysbiosis . Lactobacillus plantarum pretreatment relieved alcohol-induced subacute liver injury by reducing inflammation and enhancing antioxidative response of liver [48, 49]. We found that Lactobacillus abundance was negatively correlated with the level of phenylalanine. L. acidophilus supplementation alleviated alcohol-induced liver damage, restored IAP activity of colonic epithelial cells, and improved intestinal barrier function. Lactobacillus is an important genus that metabolizes phenylalanine, as it stimulates the expression of lactate dehydrogenase, which is responsible for the bioconversion of phenylpyruvate to phenyllactic acid . In fact, L. acidophilus supplementation enhance phenylalanine metabolism and decreased the level of phenylalanine in ALD mice, measured by bacteria functional enrichment analysis and metabolomics analysis. Although Lactobacillus was obviously negative relation with phenylalanine and its metabolites, other altered amino acids such as valine, leucine and isoleucine are also worthy of future investigation.
In this study, we reported that alcohol-related intestinal dysbiosis results in bacterial metabolism disorders of phenylalanine. Inhibition of IAP activity of colonic epithelial cells, caused intestinal inflammation and translocation of pathogen-associated molecular patterns, such as bacteria and LPS, leading to liver steatosis and injury. Supplementation with L. acidophilus restored phenylalanine metabolism and improved intestinal IAP activity, ultimately improving intestinal barrier function, and offered protection against ethanol-induced liver injury. However, we did not validate our results in a population with ALD, and this can be considered a limitation of this study.
Availability of data and materials
All data relevant to the study are included in the article and supplementary materials. More Data are available on request from the authors.
Canesso MCC, Lacerda NL, Ferreira CM, Gonçalves JL, Almeida D, Gamba C, Cassali G, Pedroso SH, Moreira C, Martins FS, Nicoli JR, Teixeira MM, Godard ALB, Vieira AT. Comparing the effects of acute alcohol consumption in germ-free and conventional mice: the role of the gut microbiota. BMC Microbiol. 2014;14:240.
Llopis M, Cassard AM, Wrzosek L, Boschat L, Bruneau A, Ferrere G, Puchois V, Martin JC, Lepage P, Le Roy T, Lefèvre L, Langelier B, Cailleux F, González-Castro AM, Rabot S, Gaudin F, Agostini H, Prévot S, Berrebi D, Ciocan D, Jousse C, Naveau S, Gérard P, Perlemuter G. Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease. Gut. 2016;65:830–9.
Leclercq S, Matamoros S, Cani PD, Neyrinck AM, Jamar F, Stärkel P, Windey K, Tremaroli V, Bäckhed F, Verbeke K, de Timary P, Delzenne NM. Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity. Proc Natl Acad Sci U S A. 2014;111:E4485–93.
Duan Y, Llorente C, Lang S, Brandl K, Chu H, Jiang L, White RC, Clarke TH, Nguyen K, Torralba M, Shao Y, Liu J, Hernandez-Morales A, Lessor L, Rahman IR, Miyamoto Y, Ly M, Gao B, Sun W, Kiesel R, Hutmacher F, Lee S, Ventura-Cots M, Bosques-Padilla F, Verna EC, Abraldes JG, Brown RS, Vargas V, Altamirano J, Caballería J, Shawcross DL, Ho SB, Louvet A, Lucey MR, Mathurin P, Garcia-Tsao G, Bataller R, Tu XM, Eckmann L, van der Donk WA, Young R, Lawley TD, Stärkel P, Pride D, Fouts DE, Schnabl B. Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease. Nature. 2019;575:505–11.
Yan AW, Fouts DE, Brandl J, Stärkel P, Torralba M, Schott E, Tsukamoto H, Nelson KE, Brenner DA, Schnabl B. Enteric dysbiosis associated with a mouse model of alcoholic liver disease. Hepatology. 2011;53(1):96–105.
Grander C, Adolph TE, Wieser V, Lowe P, Wrzosek L, Gyongyosi B, Ward DV, Grabherr F, Gerner RR, Pfister A, Enrich B, Ciocan D, Macheiner S, Mayr L, Drach M, Moser P, Moschen AR, Perlemuter G, Szabo G, Cassard AM, Tilg H. Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease. Gut. 2018;67:891–901.
Chen P, Torralba M, Tan J, Embree M, Zengler K, Stärkel P, van Pijkeren J-P, DePew J, Loomba R, Ho SB, Bajaj JS, Mutlu EA, Keshavarzian A, Tsukamoto H, Nelson KE, Fouts DE, Schnabl B. Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces ethanol-induced liver injury in mice. Gastroenterology. 2015. https://doi.org/10.1053/j.gastro.2014.09.014.
Xie G, Zhong W, Zheng X, Li Q, Qiu Y, Li H, Chen H, Zhou Z, Jia W. Chronic ethanol consumption alters mammalian gastrointestinal content metabolites. J Proteome Res. 2013;12:3297–306.
Cresci GA, Glueck B, McMullen MR, Xin W, Allende D, Nagy LE. Prophylactic tributyrin treatment mitigates chronic-binge ethanol-induced intestinal barrier and liver injury. J Gastroenterol Hepatol. 2017;32:1587–97.
Chen L, Chu H, Hu L, Li Z, Yang L, Hou X. The role of NADPH oxidase 1 in alcohol-induced oxidative stress injury of intestinal epithelial cells. Cell Biol Toxicol. 2022. https://doi.org/10.1007/s10565-022-09725-1.
Hendrikx T, Duan Y, Wang Y, Oh J-H, Alexander LM, Huang W, Stärkel P, Ho SB, Gao B, Fiehn O, Emond P, Sokol H, van Pijkeren J-P, Schnabl B. Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice. Gut. 2019;68:1504–15.
Wrzosek L, Ciocan D, Hugot C, Spatz M, Dupeux M, Houron C, Lievin-Le Moal V, Puchois V, Ferrere G, Trainel N, Mercier-Nomé F, Durand S, Kroemer G, Voican CS, Emond P, Straube M, Sokol H, Perlemuter G, Cassard A-M. Microbiota tryptophan metabolism induces aryl hydrocarbon receptor activation and improves alcohol-induced liver injury. Gut. 2021;70:1299–308.
Liu Y, Luo Y, Wang X, Luo L, Sun K, Zeng L. Gut microbiome and metabolome response of Pu-erh tea on metabolism disorder induced by chronic alcohol consumption. J Agric Food Chem. 2020;68:6615–27.
Schofield Z, Reed MA, Newsome PN, Adams DH, Günther UL, Lalor PF. Changes in human hepatic metabolism in steatosis and cirrhosis. World J Gastroenterol. 2017;23:2685–95.
Würtz P, Cook S, Wang Q, Tiainen M, Tynkkynen T, Kangas AJ, Soininen P, Laitinen J, Viikari J, Kähönen M, Lehtimäki T, Perola M, Blankenberg S, Zeller T, Männistö S, Salomaa V, Järvelin M-R, Raitakari OT, Ala-Korpela M, Leon DA. Metabolic profiling of alcohol consumption in 9778 young adults. Int J Epidemiol. 2016;45:1493–506.
Bajaj JS, Kakiyama G, Zhao D, Takei H, Fagan A, Hylemon P, Zhou H, Pandak WM, Nittono H, Fiehn O, Salzman N, Holtz M, Simpson P, Gavis EA, Heuman DM, Liu R, Kang DJ, Sikaroodi M, Gillevet PM. Continued alcohol misuse in human cirrhosis is associated with an impaired gut-liver axis. Alcohol Clin Exp Res. 2017;41:1857–65.
Lev R, Griffiths WC. Colonic and small intestinal alkaline phosphatase A histochemical and biochemical study. Gastroenterology. 1982;82:1427–35.
Lallès J-P. Recent advances in intestinal alkaline phosphatase, inflammation, and nutrition. Nutr Rev. 2019;77:710–24.
Kühn F, Adiliaghdam F, Cavallaro PM, Hamarneh SR, Tsurumi A, Hoda RS, Munoz AR, Dhole Y, Ramirez JM, Liu E, Vasan R, Liu Y, Samarbafzadeh E, Nunez RA, Farber MZ, Chopra V, Malo MS, Rahme LG, Hodin RA. Intestinal alkaline phosphatase targets the gut barrier to prevent aging. JCI Insight. 2020. https://doi.org/10.1172/jci.insight.134049.
Hamarneh SR, Kim B-M, Kaliannan K, Morrison SA, Tantillo TJ, Tao Q, Mohamed MMR, Ramirez JM, Karas A, Liu W, Hu D, Teshager A, Gul SS, Economopoulos KP, Bhan AK, Malo MS, Choi MY, Hodin RA. Intestinal alkaline phosphatase attenuates alcohol-induced hepatosteatosis in mice. Dig Dis Sci. 2017;62:2021–34.
Liu Y, Cavallaro PM, Kim B-M, Liu T, Wang H, Kühn F, Adiliaghdam F, Liu E, Vasan R, Samarbafzadeh E, Farber MZ, Li J, Xu M, Mohad V, Choi M, Hodin RA. A role for intestinal alkaline phosphatase in preventing liver fibrosis. Theranostics. 2021;11:14–26.
Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc. 2013;8:627–37.
Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, Arai H, Tanimoto I, Nishimura F, Takashiba S. Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol Med Microbiol. 2003;39:81–6.
Chu H, Duan Y, Lang S, Jiang L, Wang Y, Llorente C, Liu J, Mogavero S, Bosques-Padilla F, Abraldes JG, Vargas V, Tu XM, Yang L, Hou X, Hube B, Stärkel P, Schnabl B. The Candida albicans exotoxin candidalysin promotes alcohol-associated liver disease. J Hepatol. 2020;72:391–400.
Kim E, Yang S-M, Lim B, Park SH, Rackerby B, Kim H-Y. Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate. BMC Microbiol. 2020;20:96.
Wu H, Guang C, Zhang W, Mu W. Recent development of phenyllactic acid: physicochemical properties, biotechnological production strategies and applications. Crit Rev Biotechnol. 2021. https://doi.org/10.1080/07388551.2021.2010645.
Laursen MF, Sakanaka M, von Burg N, Mörbe U, Andersen D, Moll JM, Pekmez CT, Rivollier A, Michaelsen KF, Mølgaard C, Lind MV, Dragsted LO, Katayama T, Frandsen HL, Vinggaard AM, Bahl MI, Brix S, Agace W, Licht TR, Roager HM. Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut. Nat Microbiol. 2021;6:1367–82.
Dodd D, Spitzer MH, Van Treuren W, Merrill BD, Hryckowian AJ, Higginbottom SK, Le A, Cowan TM, Nolan GP, Fischbach MA, Sonnenburg JL. A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature. 2017;551:648–52.
Geddes K, Philpott DJ. A new role for intestinal alkaline phosphatase in gut barrier maintenance. Gastroenterology. 2008. https://doi.org/10.1053/j.gastro.2008.06.006.
Zhang J, Gong G, Wang X, Zhang H, Tian W. Positive selection on D-lactate dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus. IET Syst Biol. 2015;9:172–9.
Mu W, Yu S, Zhu L, Zhang T, Jiang B. Recent research on 3-phenyllactic acid, a broad-spectrum antimicrobial compound. Appl Microbiol Biotechnol. 2012;95:1155–63.
Liu Y, Hou Y, Wang G, Zheng X, Hao H. Gut microbial metabolites of aromatic amino acids as signals in host-microbe interplay. Trends Endocrinol Metab. 2020;31:818–34.
Suzuki R, Sato Y, Fukaya M, Suzuki D, Yoshizawa F, Sato Y. Energy metabolism profile of the effects of amino acid treatment on hepatocytes: phenylalanine and phenylpyruvate inhibit glycolysis of hepatocytes. Nutrition. 2021;82: 111042.
Yachida S, Mizutani S, Shiroma H, Shiba S, Nakajima T, Sakamoto T, Watanabe H, Masuda K, Nishimoto Y, Kubo M, Hosoda F, Rokutan H, Matsumoto M, Takamaru H, Yamada M, Matsuda T, Iwasaki M, Yamaji T, Yachida T, Soga T, Kurokawa K, Toyoda A, Ogura Y, Hayashi T, Hatakeyama M, Nakagama H, Saito Y, Fukuda S, Shibata T, Yamada T. Metagenomic and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in colorectal cancer. Nat Med. 2019;25:968–76.
Singh SB, Lin HC. Role of intestinal alkaline phosphatase in innate immunity. Biomolecules. 2021. https://doi.org/10.3390/biom11121784.
Liu W, Hu D, Huo H, Zhang W, Adiliaghdam F, Morrison S, Ramirez JM, Gul SS, Hamarneh SR, Hodin RA. Intestinal alkaline phosphatase regulates tight junction protein levels. J Am Coll Surg. 2016;222:1009–17.
Hwang SW, Kim JH, Lee C, Im JP, Kim JS. Intestinal alkaline phosphatase ameliorates experimental colitis via toll-like receptor 4-dependent pathway. Eur J Pharmacol. 2018;820:156–66.
Kaliannan K, Hamarneh SR, Economopoulos KP, Nasrin Alam S, Moaven O, Patel P, Malo NS, Ray M, Abtahi SM, Muhammad N, Raychowdhury A, Teshager A, Mohamed MMR, Moss AK, Ahmed R, Hakimian S, Narisawa S, Millán JL, Hohmann E, Warren HS, Bhan AK, Malo MS, Hodin RA. Intestinal alkaline phosphatase prevents metabolic syndrome in mice. Proc Natl Acad Sci U S A. 2013;110:7003–8.
Manna SK, Patterson AD, Yang Q, Krausz KW, Idle JR, Fornace AJ, Gonzalez FJ. UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model. J Proteome Res. 2011;10:4120–33.
Liu J, Geng W, Sun H, Liu C, Huang F, Cao J, Xia L, Zhao H, Zhai J, Li Q, Zhang X, Kuang M, Shen S, Xia Q, Wong VW-S, Yu J. Integrative metabolomic characterisation identifies altered portal vein serum metabolome contributing to human hepatocellular carcinoma. Gut. 2022;71:1203–13.
Hoyles L, Fernández-Real J-M, Federici M, Serino M, Abbott J, Charpentier J, Heymes C, Luque JL, Anthony E, Barton RH, Chilloux J, Myridakis A, Martinez-Gili L, Moreno-Navarrete JM, Benhamed F, Azalbert V, Blasco-Baque V, Puig J, Xifra G, Ricart W, Tomlinson C, Woodbridge M, Cardellini M, Davato F, Cardolini I, Porzio O, Gentileschi P, Lopez F, Foufelle F, Butcher SA, Holmes E, Nicholson JK, Postic C, Burcelin R, Dumas M-E. Molecular phenomics and metagenomics of hepatic steatosis in non-diabetic obese women. Nat Med. 2018;24:1070–80.
Gutiérrez-Díaz I, Fernández-Navarro T, Salazar N, Bartolomé B, Moreno-Arribas MV, López P, Suárez A, de Los Reyes-Gavilán CG, Gueimonde M, González S. Could fecal phenylacetic and phenylpropionic acids be used as indicators of health status? J Agric Food Chem. 2018;66:10438–46.
Kim J, Ahn SW, Kim JY, Whon TW, Lim SK, Ryu BH, Han NS, Choi H-J, Roh SW, Lee SH. Probiotic Lactobacilli ameliorate alcohol-induced hepatic damage gut microbial alteration. Front Microbiol. 2022;13: 869250.
Zhu Y, Wang X, Zhu L, Tu Y, Chen W, Gong L, Pan T, Lin H, Lin J, Sun H, Ge Y, Wei L, Guo Y, Lu C, Chen Y, Xu L. Lactobacillus rhamnosus GG combined with inosine ameliorates alcohol-induced liver injury through regulation of intestinal barrier and Treg/Th1 cells. Toxicol Appl Pharmacol. 2022;439: 115923.
Wang Y, Kirpich I, Liu Y, Ma Z, Barve S, McClain CJ, Feng W. Lactobacillus rhamnosus GG treatment potentiates intestinal hypoxia-inducible factor, promotes intestinal integrity and ameliorates alcohol-induced liver injury. Am J Pathol. 2011;179:2866–75.
Wang Y, Liu Y, Sidhu A, Ma Z, McClain C, Feng W. Lactobacillus rhamnosus GG culture supernatant ameliorates acute alcohol-induced intestinal permeability and liver injury. Am J Physiol Gastrointest Liver Physiol. 2012;303:G32–41.
Mutlu E, Keshavarzian A, Engen P, Forsyth CB, Sikaroodi M, Gillevet P. Intestinal dysbiosis: a possible mechanism of alcohol-induced endotoxemia and alcoholic steatohepatitis in rats. Alcohol Clin Exp Res. 2009;33:1836–46.
Gan Y, Chen X, Yi R, Zhao X. Antioxidative and anti-inflammatory effects of ZS62 on alcohol-induced subacute hepatic damage. Oxid Med Cell Longev. 2021;2021:7337988.
Gan Y, Chen X, Yi R, Zhao X. Antioxidative and anti-inflammatory effects of lactobacillus plantarum ZS62 on alcohol-induced subacute hepatic damage. Oxid Med Cell Longev. 2021;2021:7337988.
Cortés-Zavaleta O, López-Malo A, Hernández-Mendoza A, García HS. Antifungal activity of lactobacilli and its relationship with 3-phenyllactic acid production. Int J Food Microbiol. 2014;173:30–5.
This study was supported by the National Key R&D Program of China (No. 2022YFA1305600 to Huikuan Chu) and the Natural Science Foundation of China (No. 81974062, 81720108006 to Xiaohua Hou; No. 82000561 to Huikuan Chu).
Ethics approval and consent to participate
The animal experiments were approved by the Institutional Research Ethics Committee of the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.
Consent for publication
All authors agree for publication in this journal.
The authors declare no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Primers used in RT-PCR. Table S2. List of Lactobacillus species detected by PCR. Figure S1. Pro-inflammatory macrophages infiltration of colon tissues from mice fed ethanol and isocaloric. Colonic tissues were stained with F4/80 (1:100, Abcam) and iNOS (1:100, Arigo) by immunofluorescence according to previous reports [1, 2]. Figure S2. Lactobacillus acidophilus in stools of normal controls and alcohol exposed mice with Lactobacillus acidophilus supplement measured by qPCR. *p<0.05.
About this article
Cite this article
Chen, L., Yang, P., Hu, L. et al. Modulating phenylalanine metabolism by L. acidophilus alleviates alcohol-related liver disease through enhancing intestinal barrier function. Cell Biosci 13, 24 (2023). https://doi.org/10.1186/s13578-023-00974-z
- Alcohol-related liver diseases
- Phenylalanine metabolism
- Lactobacillus acidophilus