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

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.

1. 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, impairing oogenesis [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.