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. 2025 Oct 31;16:599. doi: 10.1186/s13287-025-04718-3

L-Glutamate enables the EGFR-MEK-ERK-mTFB2 axis to enhance mitochondrial biogenesis in intestinal stem cells

Cai-xia Dou 1, Hao-zhan Qu 1, Ying-chao Qin 1, Xiao-fan Wang 1, Jia-yi Zhou 1, Xiu-qi Wang 1, Hui-chao Yan 1,✉
PMCID: PMC12576978  PMID: 41174788

Abstract

Background

Intestinal stem cells (ISCs) sustain epithelial homeostasis through rapid mitochondrial metabolism, however, how they sense nutrient signals to regulate mitochondrial function remains unclear.

Methods

We examined the role of L-glutamate (Glu) in regulating cell mitochondrial biosynthesis using in vivo piglets, ex vivo porcine intestinal organoids (IOs), and in vitro IPEC-J2 cells.

Results

Glu enhanced jejunal development in weaned piglets. Isobaric tags for relative and absolute quantitation (iTRAQ) analysis revealed the significant enrichment of mitochondrial functions and activation of EGFR-MEK-ERK-mTFB2 signaling pathway in the jejunum. In vitro, 5 mM Glu promotes mitochondrial biosynthesis and potentiates the EGFR-MEK-ERK-mTFB2 axis. Whereas inhibition of EGFR with Osimertinib and silencing EGFR abolished these effects in IOs and IPEC-J2 cells. Colocalization and biochemical studies demonstrated interaction between Glu and EGFR in IOs.

Conclusions

Glu promotes mitochondrial biogenesis and ISC expansion by activating the EGFR–MEK–ERK–mTFB2 axis, highlighting a nutrient-sensing mechanism that couples energy availability to ISC function.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04718-3.

Highlights

1. L-Glutamate activates EGFR-MEK-ERK-mTFB2 signaling to drive mitochondrial biogenesis and intestinal stem cell (ISC) expansion.

2. Direct Glu-EGFR interaction was demonstrated in intestinal organoids, linking nutrient sensing to mitochondrial regulation.

3. Glu functions as a nutrient signal bridging energy sensing to ISC mitochondrial dynamics via EGFR.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04718-3.

Introduction

The intestinal epithelium is composed of millions of crypt-villus units. The crypt base columnar cells (CBCs), located at the base of the crypt and marked by Lgr5 (Leucine-rich repeat-containing G-protein-coupled receptor 5), represent the active intestinal stem cells (ISCs) [1]. In addition to Lgr5, ISC identity is defined by markers such as Ascl2 (Achaete scute-like 2), Olfm4 (olfactomedin 4), Msi1 (Musashi homolog 1), Smoc2, and RNF43 [2, 3]. While undergoing self-renewal, ISCs generate transit-amplifying (TA) cells, which migrate upward along the crypt-villus axis and differentiate into absorptive cells (marked by Villin), goblet cells (marked by Mucin2), and enteroendocrine cells (marked by ChgA). These differentiated cells migrate to the villus tip within 2–3 days, undergo apoptosis (as marked by caspase-3), and are subsequently shed into the intestinal lumen [4, 5]. Given that the efficient renewal of the intestinal epithelium requires substantial energy derived from mitochondrial activity [6], mitochondrial biogenesis has emerged as a critical determinant for shaping ISC fate [7, 8].

Amino acid metabolism is tightly linked to mitochondrial function. For example, amino acid deprivation reduces mitochondrial membrane potential in Drosophila, impairingoogenesis [9, 10]. Whereas amino acid excess induces the formation of mitochondrial-derived compartments (MDC) in mammalian cells for metabolic adaptation during amino acid elevation stress [11]. Mice treated with branched-chain amino acids (Leucine, Isoleucine, and Valine) exhibit enhanced mitochondrial biogenesis in skeletal muscle and hippocampus, with leucine as the most potent activator of this process [12, 13]. These findings highlight the critical role of amino acids in regulating mitochondrial biosynthesis. Although those amino acids enhance mitochondrial biogenesis in other tissues, their effects in ISCs remain unknown. As one of the most abundant dietary amino acids, L-glutamate (Glu) plays a pivotal role in intestinal health by not only serving as the primary oxidative substrate for the mucosa to produce energy but also by functioning as a signaling molecule that upregulates pathways associated with stem cell fate, thereby facilitating ISC proliferation and enhancing intestinal development [14–16]. Previous studies have demonstrated that dietary Glu enhances ISC division in Drosophila by activating metabotropic Glu receptors [17]. Similarly, dietary Glu has been shown to promote porcine ISC expansion by stimulating Frizzled7 receptors [18]. However, it remains unclear whether Glu can facilitate the proliferation of ISCs via the enhancement of mitochondrial functions.

Epidermal growth factor receptor (EGFR) is crucial for the maintenance and proliferation of ISC, and its classic upstream activation mechanism depends on ligands (such as EGF) released by Paneth cells and mesenchymal cells [19]. Additionally, EGFR activity is precisely regulated; for example, RAL GTPases activate the Drosophila intestinal MAPK signaling pathway by inducing its internalization [20]. In mouse intestinal organoids, the Cdc42 protein and its functionally enhanced splice variant, V2, play a key role in intestinal regeneration by regulating EGFR vesicular transport and binding capacity [21]. Intriguingly, studies in cancer models have revealed profound interactions between metabolic signaling and the EGFR pathway, Glu can act as a signaling molecule to induce EGFR protein stabilization via mGluR1 and as a metabolic substrate to activate the EGFR/AKT pathway through GDH1-mediated glutamine degradation in an epigenetic (KDM6A-dependent) manner, and even upregulate EGFR mRNA expression at the transcriptional level [22–24]. These findings strongly suggest that Glu may be an important upstream metabolic trigger. However, how this metabolic regulation interacts with the EGFR pathway in ISCs remains to be elucidated.

The expression of mitochondrial fusion and fission biomarkers (MFN1, MFN2, Drp1) and genes regulating mitochondrial DNA copy number (COX1, ATP6, ND1) orchestrates mitochondrial dynamics, which are essential for normal function and are ultimately supported by the mitochondrial transcription and translation machinery—initiated by mTFA, mTFB2, and POLRMT—that produces the proteins necessary for biogenesis [25–28]. Of these, mTFB2, as the primary component of the mitochondrial transcription initiation complex, plays a pivotal role in promoting both mitochondrial biogenesis and ISC proliferation through mitogen-activated protein kinase (MEK)-dependent epidermal growth factor receptor (EGFR) signaling [29, 30].

Here, we investigated how Glu regulates mitochondrial biogenesis and ISC expansion via EGFR signaling with our previous experimental strategies [30]. Using piglets as a physiologically relevant in vivo model to assess the overall nutritional impact. Utilizing intestinal organoids to specifically dissect ISC behavior and mitochondrial function in a near-native epithelial context. Employing IPEC-J2 cells as a tractable platform for detailed mechanistic interrogation of the signaling pathway.

Pharmacological inhibition of EGFR with Osimertinib and EGFR interference confirm the hypothesis that the EGFR-MEK-ERK-mTFB2 axis is the essential signaling pathway mediating Glu-induced enhancement of mitochondrial biosynthesis and ISC function.

Materials and methods

Piglet treatment

At the end of the experimental period, all of the piglets were humanely euthanised. To minimise any potential pain or distress, euthanasia was performed using a two-step procedure. First, the piglets were deeply anaesthetised via an intraperitoneal injection of pentobarbital sodium (100 mg/kg body weight) to induce irreversible unconsciousness. Following confirmation of deep anaesthesia (loss of corneal reflex), death was ensured by exsanguination. A total of sixteen weaned pigs (Duroc × Landrace × Yorkshire) with similar body weights (8.0 ± 0.5 kg) were selected and randomly assigned into two groups: a nitrogen-free diet (NFD) group and a nitrogen-free diet supplemented with 1.0% Glu (NFDG) group using a random number table (Table S1). The experiment lasted for 21 days. All samples were uniformly coded by a researcher not involved in the subsequent analyses after collection. The personnel responsible for the subsequent assays had access only to sample codes and were completely unaware of the experimental group assignments. The group information was not revealed until all data analyses were completed. As previously described [18], the proteins were extracted from the jejunal tissue of piglets and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein expression was quantified using isobaric tags for relative and absolute quantitation (iTRAQ).

Measurement of MDA and GSH-Px levels

Jejunal tissues from piglets were collected and immediately homogenized in ice-cold RIPA lysis buffer (#ES-8148, Ecotop Scientific, Guangzhou, China). The 10% tissue homogenate was centrifuged at 4 °C for 15 min, and the supernatant was carefully collected for subsequent analysis. Malondialdehyde (MDA) (#A003-1-2) and glutathione (GSH-Px) (#A005-1-2) levels in the jejunum were quantified using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s protocols.

Cell culture and treatment

IPEC-J2 cells were cultured in DMEM medium (#11965092, Gibco, NY, USA) and harvested by trypsinization (#C100C1, NCM Biotech, Suzhou, China). Based on a previously described method [31], following digestion, cells were seeded in 6-well plates (at 2 × 10⁵ cells/well) and 96-well plates (at 3 × 10³ cells/well). After undergoing amino acid starvation, the cells were divided into four treatment groups: CON, Glu (2 mM or 5 mM), Osimertinib (10, 20, 50, or 75 µM), and Glu + Osimertinib, and then cultured for 60 min.

Determination of ATP levels

IPEC-J2 cells were seeded in 6-well plates at 2 × 10⁵ cells/well. After aspiration of the original medium, 2 mL of fresh complete medium containing Glu and/or Osimertinib was added. Following 60 min of incubation, cells were washed with cold PBS and lysed with 100 µL of chilled ATP lysis buffer on a shaker for 2 min. The lysate was centrifuged at 12,000 rpm (4 °C, 5 min), and the supernatant was collected on ice. Then, 100 µL of ATP working solution was added to a white 96-well plate and incubated at room temperature for 5 min. Subsequently, 20 µL of supernatant was added to each well, and RLU was immediately measured using a microplate reader (ThermoFisher Scientific). All experiments were independently repeated three times.

Cell viability assay

IPEC-J2 cells were seeded in 96-well plates (5 × 10³ cells/well) and cultured for 24 h under standard conditions (37 °C, 5% CO₂) to allow adhesion. After treatment, 10 µL CCK-8 reagent (#C0038, Beyotime, Shanghai, China) was added to each well, followed by incubation at 37 °C for 2 h. Absorbance was then measured at 450 nm using a microplate reader (Thermo Fisher Scientific).

Intestinal organoid (IO) treatment

Following previously established protocols, jejunal crypts were isolated from piglets and subsequently cultured into IOs [32]. Then, the IOs were treated with 5 mM Glu (#G1251, Sigma-Aldrich, MO, USA) and 50 mM EGFR inhibitor Osimertinib (#HY-15772, MedChemExpress, NJ, USA) for 5 consecutive days. The growth of the organoids was monitored daily using bright-field microscopy. The organoids were then collected to detect changes in the expression of relevant proteins.

Automated capillary Western blotting (WES)

IOs were lysed with RIPA buffer (#DB258, MIKX, Shenzhen, China) for 30 min at 4℃, and the protein expression levels were quantified using WES as detailed in our previous study [18].

Western blotting

The jejunal tissues and IPEC-J2 cells were lysed with RIPA buffer. The protein concentration was normalized by dilution with distilled water. Sample lysates were separated via SDS/PAGE gel electrophoresis (#P2012, New Cell ༆ Molecular Biotech, Suzhou, China). Subsequently, the proteins were transferred onto a PVDF membrane (Millipore, Burlington). After incubation with primary and secondary antibodies (Table S2), chemiluminescent detection was performed using an ECL reagent kit (#MK-S500, MIKX, Shenzhen, China). Band density quantification was performed using ImageJ. The density value of the target protein band was divided by that of the β-actin band to obtain the target protein/ loading controls ratio. This ratio was then normalized to the average value of the CON. The data are presented as the fold change relative to the CON. All experiments were independently repeated three times.

Immunofluorescence staining (IF)

The jejunal tissues, IOs, and IPEC-J2 were fixed in 4% paraformaldehyde, sequentially dehydrated through an ethanol gradient, and permeation with xylene. Paraffin sections and IPEC-J2 were then subjected to immunostaining using primary and secondary antibodies (Table S2). Fluorescence images were acquired using an inverted fluorescence microscope (Ti2-E, Nikon, Tokyo, Japan) and analyzed with ImageJ. For each image, we randomly selected 8 fields of view for analysis. These images were captured using a 10× objective lens, with each field of view covering an actual imaging area of approximately 0.62 mm². The average fluorescence intensity values were measured based on these regions. All experiments were independently repeated three times.

Detection of reactive oxygen species (ROS) level

IPEC-J2 cells were seeded in 24-well plates and treated with 0 or 5 mM Glu. After incubating with the fluorescent probe DCFH-DA (#E004-1-1, Jiancheng Bioengineering Institute) for 1 h at 37 °C, cells were washed 3 times with PBS to remove the excess probe. Intracellular ROS generation was then assessed by measuring green fluorescence intensity using an inverted fluorescence microscope.

Real-time quantitative PCR

Total RNA was extracted from jejunal tissue using Trizol reagent, and cDNA was synthesized from 1 µg of total RNA. RT-qPCR was performed using SYBR Premix Ex Taq II (#AG11701, Accurate Biology) and analyzed using the Ct method. The primer sequences for target genes and GAPDH are provided in Table S3.

SiRNA mediated EGFR gene Silencing

IPEC-J2 cells were seeded in 12-well plates at 3 × 10⁵ cells/well in 1 mL complete medium. siRNA was designed using the DSIR (http://biodev.extra.cea.fr/DSIR/DSIR.html) and synthesized by Sangon Biotech. Before transfection, cells were washed with PBS and switched to 500 µL serum/antibiotic-free medium. For transfection complex formation, 2 µL NanoTrans™ Transfection Reagent 3000 (#CT0006, CYTOCH, Shanghai, China) was mixed with 50 µL Opti-MEM, and incubated at room temperature for 5 min. Separately, 100 µL of Opti-MEM medium was mixed with 1.25 µL (25 nM) or 2.5 µL (50 nM) of 20 µM siRNA stock solution. The two solutions were combined and incubated 15 min at room temperature. Then, 200 µL of the complex was added to each well. After 6 h, the medium was replaced with complete medium. EGFR silencing efficiency was assessed by qPCR and Western Blot after 48 h. The primer sequences are provided in Table S4.

Isothermal Titration calorimetry (ITC)

The injection syringe was used to introduce Glu (500 µmol/L, 50 µL) into the titration pool and EGFR protein (30 µmol/L, 300 µL) into the sample pool. The subsequent experimental procedures shall be conducted as previously described [33].

Pre-clinical animal studies (ARRIVE)

The work has been reported in line with the ARRIVE guidelines 2.0.

Statistical analysis

The data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 8.0 and IBM SPSS Statistics version 20.0. Comparisons between the two groups were made using the t-test. One-way analysis of variance (ANOVA) with Dunnett’s test as a post-test was used to analyze between the four groups. A significance level of P < 0.05 was adopted for all statistical tests.

Results

Dietary Glu stimulates piglet intestinal renewal through enhanced ISC activity

Weaned piglets were continuously fed either a normal-formulated diet (NFD) or NFD supplemented with 1.0% Glu (NFDG) diet for 21 consecutive days (Fig. 1A). Western blotting and IF analysis of the jejunal crypt-villus axis revealed that the expression levels of the ISC markers Lgr5 and Olfm4, proliferative cell marker Ki67, terminal differentiation marker keratin 20 (KRT20), and absorptive cell marker villin were significantly upregulated in the NFDG group compared to the NFD group (Fig. 1B-E). In contrast, the fluorescence signal intensity of the apoptotic cell marker cleaved caspase-3 (C-caspase-3) was markedly decreased in the NFDG group (Fig. 1F-G). These results indicate that dietary Glu supplementation enhances intestinal epithelial renewal and ISC activity in piglets.

Fig. 1.

Fig. 1

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Dietary 1.0% Glu stimulates piglet intestinal renewal through enhanced stem cell activity. A Schematic of the experimental design comparing the NFD and NFDG groups. B, C Protein expression levels of Lgr5, Olfm4, KRT20, and Villin in the jejunum (n = 3). D, E Representative images and statistical analysis of Ki67 staining in the jejunum (100× magnification, n = 4). F, G Representative images and statistical analysis of C-caspase3 staining in the jejunum (100× magnification, n = 4). Data are presented as mean ± SEM, *P < 0.05

Glu potentiates the mitochondrial function and EGFR-MEK-ERK-mTFB2 signaling axis in the piglet jejunal epithelium

Proteomic analysis identified 5,181 proteins, of which 48 were identified as differentially expressed between groups (24 upregulated, 24 downregulated, Fig. S1). Functional enrichment revealed significant upregulation of jejunal proteins related to intestinal development, mitochondrial biogenesis, and G protein-coupled receptors compared to the NFD group (Fig. 2A). Immunofluorescence showed strongerMFN1 and MFN2 in the NFDG group indicating accelerated mitochondrial fusion (Fig. 2B–D). Concurrently, the supplementation of Glu increased the levels of SOD1 and GSH-Px and reduced the content of MDA, indicating an improvement in the redox environment of mitochondria (Fig. S2). Although 1.0% Glu did not alter the expression of its own transporter EAAT3 (Fig. S3), Glu significantly upregulated the G protein-coupled receptor EGFR, as well as the downstream signaling cascade from cytoplasmic MEK-ERK to mitochondrial mTFB2 (Fig. 2E, -F). These findings suggest that Glu-promoted intestinal development is linked to increased mitochondrial biogenesis driven by the EGFR-MEK-ERK-mTFB2 axis.

Fig. 2.

Fig. 2

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Glu potentiates the mitochondrial function and EGFR-MEK-ERK-mTFB2 signaling axis in the piglet jejunal epithelium. A GO enrichment analysis of upregulated proteins (NFDG vs. NFD). B–D Representative images and statistical analysis of MFN1 and MFN2 in the jejunum (100× magnification, n = 4). E–F Protein expression levels of p-EGFR, p-MEK, p-ERK, and mTFB2 in the jejunum (n = 3). Data are presented as mean ± SEM, *P < 0.05

Glu facilitates EGFR-MEK-ERK-mTFB2 axis and mitochondrial function in IPEC-J2 cells

To further dissect the mechanism, we conducted a multi-parametric evaluation of mitochondrial biogenesis and functional dynamics using an established porcine intestinal epithelial (IPEC-J2) cell model. As expected, 5 mM Glu increased the proportion of PCNA⁺ mitotic cells (Fig. 3A, B), SOD1 activity and ATP levels, and decreased ROS levels (Fig. S4). Furthermore, the expression of mitochondrial biogenesis-related genes (POLRMT, mTFA, mTFB2, ATP6, COX1, and ND1) was upregulated after Glu treatment, while fusion processes (MFN1/mitofusin 1 and MFN2/mitofusin 2) were enhanced and fission (Drp1) events attenuated (Fig. 3C–G). Likewise, Glu activated the EGFR-MEK-ERK-mTFB2 signaling pathway (Fig. 3H, I). In vitro experiments reconfirmed that Glu promotes the EGFR-MEK-ERK-mTFB2 axis and mitochondrial function.

Fig. 3.

Fig. 3

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Glu facilitates EGFR-MEK-ERK-mTFB2 axis and mitochondrial function in IPEC-J2 cells. A, B Representative images and statistical analysis of PCNA in IPEC-J2 cells (200× magnification, n = 4). C–F Representative images and quantification of mitochondrial fusion/fission markers MFN1, MFN2, and Drp1 in IPEC-J2 cells (200× magnification, n = 4). G Gene abundance of POLRMT, mTFA, mTFB2, ATP6, COX1, and ND1 in IPEC-J2 cells (n = 5). H, I Protein expression levels of p-EGFR, p-MEK, p-ERK, and mTFB2 in IPEC-J2 cells (n = 4). Data are presented as mean ± SEM, *P < 0.05

EGFR inhibition abolishes Glu-induced mitochondrial remodeling via mTFB2-dependent transcriptional reprogramming

To test whether Glu directly interacts with EGFR, we started with co-localization studies in intestinal organoids (IOs). It reveals the Glu effects depend on EGFR signaling in intestinal organoids (Fig. 4A). Pharmacological inhibition of EGFR with Osimertinib (50 µM) abolished the growth advantage of intestinal organoids and IPEC-J2 cells by Glu-stimulated proliferation, as evidenced by reductions in both IO forming efficiency, budding efficiency and IPEC-J2 cell proliferation activity (Fig. S4 and Fig. 4B–D). Furthermore, Osimertinib treatment reversed Glu-triggered increase the levels of SOD1, PCNA, ATP, mitochondrial biogenesis, and mitochondrial fission (Figs. 4E and F and 5A–H and Fig.S5), and EGFR-MEK-ERK-mTFB2 cascade (Figs. 4G and H and 5I and J) in IOs and IPEC-J2 cells. Next, we conducted functional experiments by silencing EGFR (Fig. 6A–C). The results showed that Glu failed to induce the expression of mitochondrial-related markers when EGFR was inhibited (Fig. 6D, E). In addition, using purified EGFR protein from our previous study [33], we evaluated the interaction between glutamate and EGFR via isothermal titration calorimetry (ITC). The measured binding affinity (KD) was 7.561 × 10⁻⁶ M. The negative Gibbs free energy change (ΔG < 0) indicates that the binding process occurs spontaneously (Fig. 6F, G). These data collectively demonstrate that the EGFR-MEK-ERK-mTFB2 signaling circuit mediates Glu-induced mitochondrial bioenergetic regulation.

Fig. 4.

Fig. 4

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Targeting EGFR blocks Glu-driven mitochondrial biogenesis remodelling through mTFB2-dependent transcriptional reprogramming. A Colocalization of EGFR and Glu in IOs (200× magnification). B Treatment of organoids with Glu and Osimertinib (40× magnification). C, D Statistical analysis of organoid forming efficiency and budding efficiency (n = 4). E Representative images of MFN1 and MFN2 in IOs. F Gene abundance of POLRMT, mTFA, mTFB2, ATP6, COX1, and ND1 in IOs (n = 3). G, H Protein expression levels of p-EGFR, p-MEK, p-ERK, and mTFB2 in IOs (n = 3). Data are presented as mean ± SEM, *P < 0.05

Fig. 5.

Fig. 5

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Inhibition of the EGFR-MEK-ERK-mTFB2 axis abolishes the effects of Glu on mitochondrial function in IPEC-J2 cells. A, B Representative images and statistical analysis of SOD1 in IPEC-J2 cells treated with Glu and Osimertinib. C, D Representative images and statistical analysis of PCNA in IPEC-J2 cells. E–H Representative images and statistical analysis of MFN1, MFN2, and Drp1 in IPEC-J2 cells treated with Glu and Osimertinib. I, J Protein expression levels of p-EGFR, p-MEK, p-ERK, and mTFB2 in IPEC-J2 cells. Data are presented as mean ± SEM, n = 4, 200× magnification, *P < 0.05

Fig. 6.

Fig. 6

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Silencing EGFR eliminates the effect of Glu on mitochondrial function in IPEC-J2 cells. A qPCR analysis of EGFR mRNA levels in IPEC-J2 cells. B, C Western blotting analysis of EGFR in IPEC-J2 cells transfected with si-NC or si-EGFR. D, E Protein expression levels of MFN1, MFN2, and Drp1 in IPEC-J2 cells. F, G Interaction between Glu and EGFR was analyzed by ITC; (H) raw ITC data and (G) fitted curves are shown. H Model diagram of Glu-mediated regulation of ISC expansion and mitochondrial biogenesis through the EGFR-MEK-ERK-mTFB2 signaling axis. Data are presented as mean ± SEM, n = 3, *P < 0.05

Discussion

The orderly proliferation and self-renewal of ISCs are crucial for maintaining normal intestinal physiological functions [36, 37]. The rapid turnover of Lgr5+ ISCs depends on the proper functioning of mitochondria, mitochondrial dysfunction can lead to ISC loss, thereby disrupting intestinal homeostasis [36–39]. As the metabolic hub, mitochondria play a central role in amino acid metabolism and biosynthesis [40, 41]. In this study, we observed that Glu was associated with promoted development of the jejunum in weaned piglets, along with enhancements in mitochondrial biogenesis and activity along the EGFR–MEK–ERK–mTFB2 axis in both jejunal tissues and IPEC-J2 cells. Notably, inhibition of EGFR with Osimertinib abrogated the effects of Glu on MEK–ERK–mTFB2 signaling and mitochondrial biogenesis (Fig. 6H). The piglet model was selected due to its high physiological and metabolic relevance to human infants and its importance as an agricultural species. The similarities in intestinal development and amino acid metabolism between pigs and humans make piglets a valuable model for nutritional studies. However, it is important to emphasize that the observed effects of Glu are based on porcine biological systems. While the core mechanisms regulating ISC function and mitochondrial biology are often conserved across mammals, species-specific differences in nutrient metabolism, receptor expression, or signaling pathways may exist. Therefore, direct extrapolation of these results to other livestock species or humans requires caution and further validation. The pivotal role of Glu in regulating intestinal development endows it with vast potential for advancing precision nutrition strategies in swine production. These findings may also inspire more sustainable and eco-friendly livestock farming through optimized feed formulation, though applications in other species would require species-specific trials.

Amino acids, as the fundamental building blocks of proteins, play a pivotal role in regulating intestinal homeostasis and development [42]. Certain functional amino acids, acting as signaling molecules, are indispensable in modulating diverse biological processes and maintaining intestinal homeostasis. For instance, dietary supplementation with glutamine, arginine, and leucine has been shown to promote the proliferation of intestinal epithelial cells (IECs) and foster intestinal development [37, 43]. In this study, we found that dietary supplementation with 1% Glu significantly increased the proportion of ISCs and functional cells in the jejunum, simultaneously promoting epithelial growth. Moreover, crypt proliferative capacity was significantly enhanced.

Mitochondria, serving as the central hub for cellular energy production and metabolism, regulate intestinal epithelial homeostasis by controlling energy production, ROS levels, and apoptosis. Excessive ROS impairs cell proliferation and promotes cell death [44, 45]. Studies have demonstrated that HSP60 inactivation leads to mitochondrial dysfunction, resulting in diminished ATP levels and subsequently impaired self-renewal of ISCs [36]. Given that mitochondria are also primary sites of glutamate metabolism [46, 47], our findings that Glu boosts mitochondrial biogenesis and improves redox balance suggest a dual role. Although causality remains unresolved, we propose that Glu-driven redox improvements facilitate EGFR activation. EGFR is redox-sensitive: mild oxidative stress inhibits protein tyrosine phosphatases, sustaining receptor phosphorylation [48, 49]. Thus, Glu may improve redox status upstream of EGFR–MEK–ERK–mTFB2 activation, forming a positive feedback loop whereby enhanced mitochondrial function further reinforces redox homeostasis.

Previous studies established mTFB2 as a transcription factor linking EGFR–ERK signaling to mitochondrial biogenesis in Drosophila ISCs [13]. Conventionally, Glu is believed to exert its effects primarily through transporters [50]. However, in this study, the expression of EAAT3 remained unchanged in the jejunal tissue of piglets fed 1% Glu (Fig. S2). It is suggested that its promotive effect on mitochondrial biogenesis may not be primarily mediated through upregulation of this major transporter. Conversely, our data strongly support that the effect of Glu is dependent on EGFR signaling and independent of the classical uptake mechanism. Our previous research demonstrated that the G-protein-coupled receptor Frizzled7 and IR mediates Glu-induced ISC proliferation [18, 31]. Therefore, we propose that EGFR senses Glu stimulation and amplifies ERK-MEK-ERK-mTFB2 signaling, thereby enhancing mitochondrial biogenesis and promoting ISC expansion. Moreover, the application of Osimertinib further confirmed the pivotal role of EGFR signaling in Glu-mediated regulation of mitochondrial biogenesis and ISC expansion.

In summary, Glu enhances mitochondrial biogenesis and ISC expansion by potentiating EGFR-MEK-ERK-mTFB2 signaling, thereby promoting intestinal epithelial renewal. These findings identify Glu as a key nutrient–signaling molecule linking energy sensing with ISC mitochondrial dynamics and intestinal homeostasis.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1. (807.8KB, docx)

Acknowledgements

The authors would like to gratefully appreciate the National Key Research and Development Program of China (2023YFE0124400) and the Science and Technology Planning Project of Guangzhou (2024B03J1267). AI was not used to design or write this manuscript.

Artificial intelligence (AI)

The authors declare that they have not use AI-generated work in this manuscript.

Author contributions

D.C.: conceptualization, performing experiments, original draft writing. Q.H.: data curation. Q.Y.: performing experiments. W.X.: supervision. Z.J.: writing-reviewing and editing. W.X.: conceptualization, and funding acquisition. Y.H.: conceptualization, design, and editing.

Funding

This research was funded by the National Key Research and Development Program of China (2023YFE0124400), and the Science and Technology Planning Project of Guangzhou (2024B03J1267).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All experimental piglets were humanely euthanized in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC). Approved project: Molecular network reveal of glutamate regulating small intestinal stem cell development in piglets based on organoid model. The institutional approval unit: South China Agricultural University. Approval number: 2023F086. Date of approval:2023-03-06.

Consent for publication

That it is not under consideration for publication elsewhere.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (807.8KB, docx)

Data Availability Statement

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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