Molecular and Cellular Biology

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in regulating cellular senescence and aging. Our recent studies identified a conserved C. elegans miRNA cluster (miR-229/64/65/66) that is required for normal adult lifespan, with overexpression significantly extending longevity. Notably, cel-miR-229 is evolutionarily conserved in humans, with hsa-miR-425 sharing an identical seed sequence. Here, we investigated the role of miR-425 in mammalian cellular senescence. We found that miR-425 expression is markedly reduced in pharmacologically induced senescence in human lung cancer cells. Restoration of miR-425 expression attenuates senescence and suppresses the expression of senescence-associated secretory phenotype (SASP) cytokines following senescence induction. We further observed that miR-425 levels decline during replicative senescence, whereas stable overexpression in WI-38 fibroblasts delays senescence accumulation and preserves proliferative capacity. Mechanistically, miR-425 suppresses TGF-{beta} signaling, leading to reduced expression of the cyclin-dependent kinase inhibitor p21/CDKN1A and increased phosphorylation of the retinoblastoma (RB) protein, thereby promoting cell-cycle progression. We further identify PPP2CB, the catalytic subunit of protein phosphatase 2A (PP2A), as a direct target of miR-425. PPP2CB expression is downregulated in miR-425-5p overexpressing cells, even under senescence induction. Knockdown of PPP2CB using siRNA phenocopies the effects of miR-425 overexpression, reducing senescence, enhancing proliferative potential, and increasing RB phosphorylation. Collectively, our findings identify miR-425 as a conserved regulator of cellular senescence that acts through upregulation of RB phosphorylation. These results establish a novel miR-425-PPP2CB-RB regulatory axis controlling proliferation and senescence and suggest miR-425 as a potential therapeutic target for mitigating senescence to promote extended health span.

HSP90/R2TP is an essential quaternary chaperone composed of RPAP3, PIH1D1 and the RUVBL1/RUVBL2 AAA+ ATPases. These enzymes are also part of the INO80, SRCAP and TIP60 complexes, but the relationship between these chromatin remodelers and R2TP remains unclear. Here, we performed systematic analyses of the R2TP-specific subunits RPAP3 and PIH1D1. We validated 115 interaction partners and found that many were sensitive to HSP90 or R2TP inhibition. In yeast, epistatic screens revealed functional interactions with Ino80, Swr1 (SRCAP) and NuA4 (TIP60). Consistently, human RPAP3 physically interacted with subunits of INO80, SRCAP and TIP60 and was required for the formation of these complexes. More specifically, RPAP3 enabled the co-translational association of RUVBL1/RUVBL2 with the motor subunit of these chromatin remodelers. In vitro, the client-binding domain of RUVBL1/RUVBL2 modulated their interaction with RPAP3, suggesting that client subunits displace RPAP3 from nascent complexes. Thus, R2TP is an early chaperone of TIP60, SRCAP and INO80, which leaves RUVBL1/RUVBL2 as resident scaffolding subunits.

The primary cilium has been implicated in multiple developmental processes, such as cell migration and asymmetric cell division of stem- and progenitor cells. While most in vitro model systems examine ciliogenesis induced by serum starvation, it is not fully understood how de- and re-ciliation are regulated in proliferating stem- and progenitor cells. Here we employ the hierarchically organized C2C12 skeletal muscle cell line to examine how K-Ras4B participates in de- and re-ciliation processes of ciliated stem- and progenitor cells. We show that MAPK-pathway activation supports ciliogenesis through phosphorylation of centrosomal protein CEP55, which can then no longer stabilize the master regulator of de-ciliation Aurora kinase A. K-Ras4B localizes to the primary cilium aided by the ciliary trafficking chaperone PDE6D, which promotes ciliation. In line with this, depletion of components of the PDE6D machinery, RPGR and RPGRIP1L, decreases ciliation. Activation of the ciliary AMPK-PKG2-pathway increases S181-phosphorylation of K-Ras4B, which negatively regulates its binding to PDE6D, its ciliary abundance and promotes differentiation. Our work integrates a major mediator of mitogenic signaling into the regulation of ciliogenesis of proliferating muscle stem- and progenitor cells.

Skeletal muscle homeostasis depends on the coordinated regulation of protein turnover and mitochondrial quality control; however, the molecular mechanisms linking these processes remain unclear. In this study, we examined the physiological role of leucine aminopeptidase 3 (LAP3), a post-proteolytic aminopeptidase, using constitutive LAP3-deficient mice. LAP3 deficiency preferentially affected skeletal muscle, causing reduced muscle mass and mitochondrial enlargement in both sexes. Female LAP3-deficient mice also showed reduced myofiber size, impaired endurance capacity, increased energy expenditure, elevated lipid oxidation, and lipid droplet accumulation adjacent to the mitochondria. Proteomic analyses revealed remodeling of pathways related to lipid metabolism and protein homeostasis. Consistent with these findings, LAP3 deficiency increased the expression of Pink1 and Tax1bp1 and promoted the accumulation of ubiquitinated proteins, suggesting alterations in mitochondrial quality control and proteostatic regulation. In cultured myogenic cells, LAP3 localized to mitochondrial fractions, and both LAP3 knockdown and overexpression altered mitochondrial morphology. Taken together, these results identify LAP3 as a regulator of skeletal muscle homeostasis and support a role for LAP3 in linking intracellular peptide turnover to mitochondrial homeostasis, with female skeletal muscle showing greater susceptibility to LAP3 deficiency.

Understanding which genes are involved in mutagenesis is essential for developing cancer prevention and treatment strategies; establishing protectors of the genome has revolutionized cancer biology. Here, we describe metallothionein (MT) proteins as previously uncharacterized protectors against mutagenesis. MT is a heavy metal binding protein essential for zinc homeostasis and protection against heavy metal cytotoxicity. Because zinc binds approximately 10-15% of the proteome and is critical for processes such as DNA repair and mitochondrial health, MT loss is expected to disrupt these processes. We hypothesized that MT loss induces genomic instability by impairing DNA repair and mitochondrial function. In this study, the consequences of MT deficiency in high-grade serous ovarian cancer (HGSC) were investigated by knockdown of the most highly expressed MT, MT2A. Loss of MT2A resulted in the impaired DNA repair pathway base excision repair (BER), leading to increased mutagenesis. MT2A deficiency produced mitochondrial dysfunction, characterized by a decrease in mitochondrial membrane potential, glycolysis, oxidative phosphorylation, amino acids, and an imbalance of nucleobases. Together, these defects reflect cellular states associated with increased cancer aggressiveness. These findings identify MT as a fundamental hub maintaining genomic and metabolic integrity.

Premature termination of transcription (PTT), also known as attenuation, is a conserved gene regulatory mechanism that operates across all domains of life and in viruses. Attenuation enables rapid cellular responses to environmental and metabolic changes and fine-tunes expression of biosynthetic genes. In Saccharomyces cerevisiae, attenuation of RNA Polymerase II (Pol II) transcription was first linked to the Nrd1-Nab3-Sen1 (NNS) termination pathway for non-coding RNAs, and the mRNA 3-end processing factor Hrp1 has been implicated more recently. Substitutions in Hrp1 RNA Recognition Motifs (RRMs) cause attenuator readthrough and reduce RNA-binding affinity in vitro, but direct evidence for Hrp1 functioning at attenuators in vivo remains limited. Here, we characterized 5-end RNA terminator elements from several genes, including RAD3, SNG1, MNR2, and CPR8. Readthrough mutations clustered in AU-rich regions resembling polyadenylation site (pA) efficiency elements, consistent with Hrp1 binding targets. Amino acid substitutions of Hrp1 RRM residue F162 revealed a general requirement for aromaticity in RNA recognition that varied to some degree by gene context. To test Hrp1-RNA interactions independent of other yeast factors, we adapted a bacterial 3-hybrid (B3H) assay. Hrp1 interacted with RNA derived from the GAL7 3-end pA site and 5-end terminator regions of RAD3, MNR2, and CPR8. Mutations in AU-rich RNA regions that disrupted Pol II attenuation in yeast generally impaired B3H interactions. However, some Hrp1 mutants (M191T, I270T, D271G, M275V, T280I) retained binding to CPR8 terminator RNA, suggesting their defects require additional yeast components. These results demonstrate that Hrp1 is sufficient to bind multiple UA-rich attenuator RNAs in vivo, expanding Hrp1 function to include early transcription events.

Stem cells proliferate rapidly to maintain fast tissue turnover during regeneration. However, the feedback mechanisms in stem cells that prevent hyperproliferation remain unclear, and their dysregulation can lead to organ failure and cancer. Here, we identified nuclear factor-Y (NF-Y) as the transcriptional repressors to maintain the stem cell quiescence during intestinal homeostasis. We found that NF-Y negatively regulates intestinal stem cell (ISC) proliferation through preferentially occupying the promoters of EGFR signaling pathway components Egfr/Mkp3/Raf/Ras/pointed, via the action of histone acetyltransferase Nejire (Nej)/p300 dependent transcription regulation. While the loss of NF-Y enhances ISC proliferation, cell death and sensitivity to stress and tumor induced mortality. Moreover, NF-Y acts together with Nej to restrict Egfr expression and suppress ISC hyperproliferation. Together, these results demonstrate NF-Y acts with Nej serve as a key negative feedback module to orchestrate transcription initiation and termination of growth signaling in the control of stem cell activity in homeostatic and disease conditions.

Dietary methionine restriction has been shown to improve metabolic health and treat multiple diseases. Methionine metabolism regulates transmethylation reactions, including N6-methyladenosine (m6A) RNA methylation, by modulating the availability of S-adenosyl methionine (SAM). Both m6A RNA methylation and methionine metabolism are involved in the regulation of the circadian clock. However, it remains unclear whether dietary methionine influences circadian rhythms through the regulation of m6A RNA modification. In this study, we investigated the effects of short-term methionine deprivation on the diurnal oscillations of m6A RNA methylation in the mouse liver. We found that a methionine-deficient (MD) diet reprogrammed the cyclic expression patterns of m6A writers, erasers, and readers. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that the MD diet induced de novo diurnal m6A oscillations in genes associated with RNA processing, protein translation, protein ubiquitination, and mTORC1 signaling pathways. RNA-seq and quantitative proteomics analyses demonstrated that MD-induced changes in m6A RNA levels were linked to alterations in mRNA and protein abundance. We observed that dynamic m6A RNA methylation of the transcripts encoding two key enzymes, MAT2A and CBS, helps maintain methionine homeostasis in response to methionine starvation. These findings identify m6A RNA methylation as a key mechanism linking methionine metabolism to circadian regulation.

Histone proteins, which reside in the nuclei of eukaryotic cells, are involved in diverse cellular processes. The core histone H4 serves as a structural component of the nucleosome. Patients carrying mutations in H4Clustered histone (H4C) genes exhibit a broad spectrum of developmental abnormalities, including short stature, microcephaly, intellectual disability, growth retardation, and digital anomalies. However, the impact of H4 mutations on mammalian embryogenesis remains largely unclear. Here, we demonstrate that histone H4C genes play crucial roles in skeletal development and cortical neurogenesis. We found that mRNAs of the histone H4C gene family are specifically expressed in proliferating progenitor cells in the developing mouse neocortex and in human induced pluripotent stem cell-derived cortical organoids. CRISPR-mediated disruption of H4C3 in mice caused severe defects in skeletal formation and neocortical neurogenesis. Furthermore, overexpression of a mutant form of H4C3 resulted in altered expression of genes associated with cellular migration and motility. Together, these findings suggest that histone H4 plays a critical role in regulating the balance between proliferation and differentiation during mammalian embryonic development, thereby explaining the broad spectrum of patient phenotypes.

p53-binding protein 1 (53BP1) is a key mediator of the DNA damage response and genome stability. While its interphase function is well-characterized, its mitotic role remains less understood. Here we show that aberrant activation of 53BP1 through the loss of its negative regulator, TIRR, leads to mitotic abnormalities including altered spindle geometry, kinetochore-microtubule (k-MT) attachment errors and whole chromosome missegregation. We demonstrate that loss of TIRR results in excess interaction between 53BP1 and the key mitotic kinase Polo-like kinase 1 (PLK1), altering PLK1s activation, spatial distribution, and its interaction with known PLK1 substrates at multiple mitotic stages. Moreover, due to PLK1s established role in CENP-A loading, hyperactivation of 53BP1 compromises CENP-A loading, triggers gradual loss of CENP-A from centromeres and generates severe kinetochore assembly defects. These findings uncover a non-canonical mitotic function of 53BP1 as a key regulator of PLK1 activity and chromosome segregation fidelity.

IDH1 R132 mutations are among the most frequent hotspot mutations in cancer, but their mutational origins have remained unclear. Here, we provide evidence that IDH1 R132C, the predominant IDH1 mutation in cholangiocarcinoma, acute myeloid leukemia, and melanoma, likely arises through APOBEC3-mediated mutagenesis. IDH1 R132C is a TpC>TpT substitution on the lagging-strand DNA template within a hairpin-forming sequence context, consistent with APOBEC3 susceptibility. In vitro assays showed that APOBEC3A can deaminate the relevant cytosine, and APOBEC3A and APOBEC3B were relatively highly expressed in tumor types with recurrent IDH1 R132C mutations. IDH1 R132G, a TpC>TpG substitution at the same site, may similarly result from APOBEC3 activity. By contrast, IDH1 R132H, the predominant IDH1 mutation in lower grade glioma and glioblastoma, is a CpG>TpG substitution at a methylated cytosine on the leading-strand DNA template, a pattern more consistent with DNA polymerase epsilon replication error. Concordantly, tumor types enriched for IDH1 R132H showed relatively low POLE expression. Together, these in vitro and bioinformatic analyses provide insight into the distinct mutational mechanisms that likely underlie recurrent IDH1 hotspot mutations in cancer.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

TP53 (also called TRP53 or p53) is a critical tumour suppressor that prevents cancer development by inducing a transcriptional program which can lead to diverse cellular responses, most prominently, cell proliferation arrest/senescence with survival of cells or cell death by apoptosis. Why distinct cell types undergo different outcomes after p53 activation remains unclear. Using integrated RNA-sequencing, proteomic and functional analyses across a diverse range of murine primary cell types, we demonstrate that cell fate is governed by the balance between pro-survival BCL-2 and pro-apoptotic BH3-only proteins. Cells resistant to apoptosis displays a higher starting ratio of pro-survival BCL-2 to pro-apoptotic BH3-only proteins, along with transcriptional upregulation of the pro-survival gene Bcl2l1, encoding BCL-XL. This control of cell fate is also seen in human wild-type p53 cancer cell lines. These findings reveal the mechanism for understanding p53-driven cell fate decisions, suggest therapeutic strategies to shift p53-induced cell proliferation arrest/senescence toward apoptotic cell death and allowed generation of an RNAseq data-based predictor of outcome for cancer cells after p53 activation.

SMAD4 is a central transcriptional effector of the TGF-{beta} signaling pathway and a frequently inactivated tumor suppressor gene in various cancers. Missense mutations in its MH2 domain are among the most prevalent somatic alterations in colorectal cancer (CRC). These mutations are associated with disease progression and poor prognosis, yet their precise mechanistic consequences have remained incompletely characterized. Here, we show that CRC-derived SMAD4 MH2 hotspot mutations (D351H, S357P, R361C, and R361H) selectively impair co-activator recruitment without disrupting chromatin occupancy. RNA-seq profiling demonstrated broad suppression of TGF-{beta} target gene expression across all mutants. Notably, the mutations confer distinct degrees of TGF-{beta} pathway unresponsiveness: R361H is completely refractory to TGF-{beta} stimulation, whereas R361C and S357P retain partial transcriptional responsiveness suggesting allele-specific differences in the severity of co-activator interface disruption. Genome-wide chromatin binding analysis by greenCUT&RUN confirmed that all mutants maintain wild-type-like genomic occupancy, as expected given that the MH1 DNA-binding domain is intact in each case. Proximity-dependent biotinylation mass spectrometry in COLO205 cells revealed that all four mutants exhibit markedly reduced interactions with the CREBBP/EP300 histone acetyltransferase complex and BRD4 relative to wild-type SMAD4 identifying disrupted co-activator engagement. Collectively, our findings establish that SMAD4 MH2 mutations impair TGF-{beta}-induced transcription by selectively reducing CREBBP/EP300 recruitment, which provides a molecular mechanism for the loss-of-function SMAD4 phenotype in CRC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/735541v1_ufig1.gif" ALT="Figure 1000"> View larger version (24K): org.highwire.dtl.DTLVardef@14f542eorg.highwire.dtl.DTLVardef@11fd220org.highwire.dtl.DTLVardef@1c3aa1org.highwire.dtl.DTLVardef@14d5a8e_HPS_FORMAT_FIGEXP M_FIG C_FIG

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

Erythroferrone (ERFE) is an erythroblast-secreted hormone that suppresses hepatic hepcidin expression to increase iron availability for erythropoiesis, ensuring recovery from anaemia. ERFE excess drives iron overload in disorders of ineffective erythropoiesis. Despite its pivotal role in systemic iron homeostasis and diseases of erythropoiesis, ERFEs molecular regulation has remained undefined. Here, we applied a genomic approach to characterise the molecular mechanisms governing ERFE expression. Using the HUDEP-2 human erythroid progenitor model, integrative ATAC-seq, CUT&RUN and micro capture-C analysis we identified a stage-specific accessible chromatin region within the ERFE 3 UTR that interacts with the promotor. We also identified enhancer-associated chromatin marks including H3K4me1 and H3K27ac in this region, and demonstrate that this cis-regulatory element is bound by key erythroid transcription factors KLF1, GATA1, TAL1 and STAT5. Functional dissection using CRISPR-Cas9-mediated deletion of the central 3 UTR enhancer element led to marked reduction in ERFE mRNA expression, and we show a corresponding reduction in nascent mRNA, confirming a key role for this region in transcriptional regulation. We define the transcriptional regulatory mechanism by which maturing human erythroblasts activate ERFE, the endocrine signal that coordinates erythropoietic demand with systemic iron mobilisation.

The developing spinal cord contains both neural and non-neural tissues that arise within a shared morphogen signaling environment, raising the question of how distinct lineage identities are established and maintained. A striking example of this is the floor plate (FP), a mesoderm-derived, non-neural midline structure that functions as a critical signaling center adjacent to neural progenitor domains. Here, using zebrafish, we show that the pioneer transcription factor foxa2 is expressed in the FP, adjacent p3 neural progenitors, and p3-derived Kolmer-Agduhr" (KA") interneurons. Loss of foxa2 results in a complete loss of canonical FP identity and an expansion of p3 progenitors. Lineage tracing reveals that, in the absence of foxa2, FP cells undergo a fate transformation into neuron-producing p3-like cells, indicating that Foxa2 functions as a lineage barrier to preserve non-neural FP identity. In contrast, within the neural lineage, loss of foxa2 leads to elevated Sonic hedgehog (Shh) pathway activity and impaired KA" differentiation, suggesting that Foxa2 negatively regulates Shh responsiveness. Conversely, Foxa2 overexpression induces ectopic FP and KA" marker expression in a stage-dependent manner. Together, our findings reveal a dual role for Foxa2 in maintaining the non-neural FP lineage while fine-tuning morphogen responsiveness in neighboring neural progenitors during spinal cord development.

Processing of the precursor ribosomal RNAs (pre-rRNAs) is a key aspect of ribosomal subunit assembly that is closely coordinated with other maturation events. The ribonucleases that mediate pre-rRNA cleavages require regulation to ensure that their activities are exerted in a timely manner. Post-translational modifications can influence protein functions, and although many human ribosome assembly factors are reported to be post-translationally modified, most of these sites remain unconfirmed and functional insights are lacking. Here, we show that NOB1, the PIN domain endoribonuclease responsible for cleavage of the 3' end of the 18S rRNA, is phosphorylated within an evolutionarily conserved acidic tract that can be modified by casein kinase II in vitro. Association of NOB1 with pre-ribosomes is independent of these phosphorylations, and lack of NOB1 phosphorylation only mildly perturbs the efficiency of SSU maturation events upstream of 3' end cleavage of the 18S rRNA. Interestingly, our analyses of pre-rRNA levels in cells depleted of NOB1 or lacking its catalytic activity revealed not only accumulation of the 18SE precursor of the 18S rRNA, but also altered levels of pre-rRNAs containing 5' external transcribed spacer (ETS) sequences (43S, 26S and 30S). This suggests that lack of NOB1-mediated pre-rRNA cleavage impairs recycling of assembly factors required during early biogenesis steps, leading to altered kinetics of 5' ETS processing. Taken together these data provide new insights into the role of NOB1 during SSU biogenesis and the post-translational regulation of this ribonuclease.

WNT11, a non-canonical WNT ligand, plays well-defined roles in development and tissue architecture; however, its function in cancer remains ambiguous. Here, we characterize WNT11 as a context-dependent suppressor of cancer stemness, invasion, and in vivo tumor formation in human epithelial cancer models. We show that WNT11 upregulation reduces the expression of stemness-promoting genes, suppresses epithelial-to-mesenchymal transition, and inhibits sphere formation and tumor growth. Conversely, downregulation of WNT11 enhances these aggressive malignant properties of cancer cells. Mechanistically, we found that the ability of WNT11 to inhibit RAC1 GTPase activation is essential for its regulation of invasion and self-renewal. In cells unresponsive to WNT11, the connectivity between WNT11 and RAC1 activity is disengaged. Direct manipulation of RAC1 activity in these cells recapitulates the phenotype and molecular signature of WNT11-responsive cells, establishing RAC1 as a critical effector of WNT11-mediated tumor suppression. Taken together, these findings identify the cellular context in which WNT11 suppresses RAC1 activation as a key determinant of its anti-tumor effects and provide a mechanistic framework for understanding the diverse, and sometimes opposing, roles of WNT11 reported in cancer.

The liver- cell axis is a finely tuned biological rheostat that regulates whole body amino acid availability. Pancreatic cells secrete glucagon that regulates amino acid catabolism through gluconeogenesis and ureagenesis, yet the mechanisms linking amino acid levels to cell growth and function are not fully understood. Here, we identify glutaminase, the enzyme that catalyzes glutamine catabolism, as a critical cell regulator. Glutaminase is highly enriched in cells across species. cell expression of glutaminase is required for nutrient-dependent mTORC1 activation, suppression of AMPK signaling, and sustained expression of the glutamine transporter SLC38A5. This establishes a feed-forward loop linking glutamine metabolism to amino acid sensing and growth. Reduced glutaminase activity impairs dynamic glucagon secretion in response to low glucose and amino acids. Together, these findings highlight the importance of glutamine metabolism in cell growth and hormone secretion and suggest it may play a role in cell adaptation to hyperaminoacidemia.