Lactobacillus salivarius metabolite succinate enhances chicken intestinal stem cell activities via the SUCNR1-mitochondria axis

Abstract

The activity of intestinal stem cells (ISCs) can be modulated by Lactobacillus, which subsequently affects the mucosal absorptive capacity. However, the underlying mechanisms remain unclear. In this study, a total of 189 Hy-Line Brown chickens (Gallus) were randomly assigned to one of seven experimental groups (n = 27 per group). These groups included a control group, a vehicle group (MRS group), a Lactobacillus salivarius group, a L. salivarius supernatant group, and three succinate treatment groups with various dosages. Each group was further subdivided into three replicates, with 9 chickens per replicate. The results indicate that the administration of Lactobacillus salivarius supernatant to laying hens notably increased the mRNA abundance of the amino acid transporters oligopeptide transporter 1 (PepT1) and sodium-dependent neutral amino acid transporter (B⁰AT). Metabolomic analyses indicated that the supernatant contains a high concentration of organic acids. Among them, succinate could enhance mRNA abundance of PepT1, B⁰AT and excitatory amino acid transporters 3 (EAAT3) both in vivo and in vitro. Accordingly, succinate could accelerate intestinal epithelial turnover, as indicated by the increased levels of cyclin-dependent kinase 2 (Cdk2) mRNA and proliferating cell nuclear antigen protein (PCNA), as well as ISC differentiation-related protein leucine-rich repeat containing G protein-coupled receptor 5 (LGR5). Furthermore, succinate treatment was shown to elevate the levels of mitochondrial fusion proteins optic atrophy 1 (OPA1) and translocase of outer mitochondrial membrane 20 (TOMM20), resulting in increased local ATP levels. However, pretreatment with NF-56-EJ40, a succinate receptor antagonist, attenuated the effects of succinate on OPA1, TOMM20, and ATP levels, alone with the reducing LGR5 and PCNA levels. Collectively, succinate, a metabolite of L. salivarius, activates the SUCNR1-mitochondria axis in ISCs, facilitating mitochondrial ATP synthesis, promoting ISC activity, and ultimately enhancing mucosal absorptive capacity.

Keywords: Intestinal stem cell; Lactobacillus salivarius; Mitochondria; Succinate.

Figure 1

The supernatant of L. salivarius improved intestinal mucosal absorptive capacity and host's intestinal flora. Laying hens were fed the supernatant of L. salivarius for 14 consecutive days. (A-C) Histograms represent the alterations in mRNA abundance of amino acid transporters PepT1, B⁰AT, and EAAT3 in the jejunum. The data is presented as mean ± standard deviation (n = 6). Columns without common letters indicate significant differences (P-value < 0.05) among the various treatments. (D) Cluster heatmaps of species abundance for the top 35 at the phylum level. (E-F) Histograms and cladograms represent the values of linear discriminant analysis (LDA). Con, chickens were fed a basal diet; MRS, as vehicle group, chickens were fed a basal diet containing MRS; L. sa., chickens were fed a basal diet containing L. salivarius; L. sa. s, chickens were fed a basal diet containing L. salivarius supernatant.

Figure 2

Screening for the essential metabolite in the L. salivarius supernatant regulates intestinal mucosal absorptive capacity. Untargeted metabolomics analysis was conducted on the L. salivarius supernatant, with MRS used as the vehicle group. (A, C) The PCA results in both negative and positive polarity modes. (B, D) The PLSDA results in both negative and positive polarity modes. (E) Hierarchical clustering analysis of differential metabolites between the MRS and the L. salivarius supernatant.

Figure 3

Screening effective organic acids and determining their optimal dosage to enhance intestinal mucosal absorptive capacity. Histograms represent the alterations in mRNA abundance of amino acid transporters in the enteroids treated with Succinate (A-C), Malonic acid (D-F), or Folic acid (G-I) at dosages ranging from 0.1 mM to 10 mM, respectively, for 48 h. (J-L) Histograms represent the alterations in mRNA abundance of PepT1, EAAT3 and Tnf-α in the jejunum of laying hens fed succinate at dosages ranging from 5 to 20 mg/kg BW. (M) Histograms represent the alterations in mRNA abundance of Tnf-α, Il-1β, and Il-6 in enteroids treated with 1 mM succinate. (N) The daily egg production rate of each experimental group in vivo. (O) The height of eggs albumen from each experimental group in vivo. (P) The Haugh unit of eggs from each experimental group in vivo. The data is presented as mean ± SEM. ns, no significant difference compared to the control group, * P-value < 0.05 compared to the control group, ** P-value < 0.01 compared to the control group.

Figure 4

Succinate promotes intestinal epithelial turnover and ISC activities. (A) Histogram represents the alterations in mRNA abundance of Cdk2, Bax, and Lgr5 in enteroids treated with 1 mM succinate. (B, C) Western blot analysis of LGR5 and PCNA proteins in enteroids treated with 1 mM succinate. (D) The immunofluorescent staining of LGR5 protein in enteroids, scale bar = 10 μm. (E-G) Western blot assay of LGR5 and PCNA proteins in the intestinal crypts of laying hens fed with succinate at varying dosages. The data is presented as mean ± standard deviation (n = 3). ns, no significant difference compared to the control group, * P-value < 0.05 compared to the control group, ** P-value < 0.01 compared to the control group.

Figure 5

SUCNR1 mediates the regulation of succinate on ISC activity. The enteroids were pre-treated with the SUCNR1 antagonist NF-56-EJ40 for 30 min, followed by 1 mM succinate treatment for 48 h. (A-C) Western blot assay of LGR5 and PCNA proteins in enteroids. (D-G) Western blot analysis of OPA1 and TOMM20 proteins in enteroids. (H-J) Western blot assay of OPA1 and TOMM20 proteins in the intestinal crypts of hens that were fed with succinate at varying dosages. (K) The immunofluorescent co-staining of LGR5 and TOMM20 in enteroids, scale bar = 50 μm or 100 μm. The data is presented as mean ± standard deviation (n = 3). Columns without common letters indicate significant differences (P-value < 0.05) among various groups.

Figure 6

Screening of downstream signaling pathways activated by SUCNR1 in response to succinate. RNA sequencing was performed on enteroids pretreated with an antagonist NF-56-EJ40 followed by succinate treatment. (A-B) The volcano plot displays the differentially expressed genes between the succinate group and the control groups, as well as between the succinate + NF-56-EJ40 group and the succinate group. SN, succinate + NF-56-EJ40. (C-D) KEGG and GO analysis revealed enriched pathways between the succinate + NF-56-EJ40 group and the succinate group. (E) Hierarchical clustering analysis of differentially expressed genes among various groups. (F) Directed Acyclic Graph assay illustrates the functions of the differentially expressed genes. N, NF-56-EJ40. S + N, succinate + NF-56-EJ40.

Figure 7

SUCNR1 mediates the effect of succinate on the glycometabolism-related pathway. The enteroids were pretreated with NF-56-EJ40 (SUCNR1 antagonist) followed by succinate treatment. (A-F) Histograms represent mRNA abundance alterations of Wnt3a/β-catenin-related genes (Wnt3a, β-catenin), OXPHOS-related enzymes (Sdha, Cox8a), desuccinylation-related enzyme (Sirt5), and succinylation-related enzyme (Suclg2) in enteroids. (G-J) Western blot assay of SDHA, WNT1, and COX5B proteins in enteroids. The data is presented as mean ± standard deviation. Columns without common letters indicate significant differences among the treatments (P < 0.05), n = 3.

Figure 8

Schematic representations of the main findings in this work. Succinate, a metabolite of L. salivarius, plays a crucial role in activating the SUCNR1-mitochondria axis in ISC, which further promotes mitochondrial fusion, ATP synthesis, and enhances ISC differentiation and proliferation. Ultimately, this process leads to accelerated turnover of the epithelium, enhancing mucosal absorptive capacity.