Intestinal tryptophan metabolism in disease prevention and swine production.

Tryptophan (Trp) is an essential amino acid that cannot be synthesized by animals. It has been characterized into two different isomers, levorotation-Trp (L-Trp) and dextrorotation-Trp (D-Trp), based on their distinct molecule orientation. Intestinal epithelial cells and gut microbiota are involved in metabolizing L-Trp in the gut via the activation of the kynurenine, serotonin, and indole pathways. However, knowledge regarding D-Trp metabolism in the gut remains unclear. In this review, we briefly update the current understanding of intestinal L/D-Trp metabolism and the function of their metabolites in modulating the gut physiology and diseases. Finally, we summarize the effects of Trp nutrition on swine production at different stages, including growth performance in weaned piglets and growing pigs, as well as the reproduction performance in sows.

Glutamine boosts intestinal stem cell-mediated small intestinal epithelial development during early weaning: Involvement of WNT signaling.

Early weaning usually causes small intestine epithelial development abnormality, increasing the risk of gastrointestinal diseases. Glutamine (Gln), enriching in plasma and milk, is widely reported to benefit intestinal health. However, whether Gln affects intestinal stem cell (ISC) activity in response to early weaning is unclear. Here, both the early weaning mice and intestinal organoids were used to study the role of Gln in regulating ISC activities. Results showed that Gln ameliorated early weaning-induced epithelial atrophy and augmented the ISC-mediated epithelial regeneration. Gln deprivation disabled ISC-mediated epithelial regeneration and crypt fission in vitro. Mechanistically, Gln augmented WNT signaling in a dose-dependent manner to regulate ISC activity, while WNT signaling blockage abolished the effects of Gln on ISCs. Together, Gln accelerates stem cell-mediated intestinal epithelial development associated with the augmentation of WNT signaling, which provides novel insights into the mechanism by which Gln promotes intestinal health.

Early weaning causes small intestinal atrophy by inhibiting the activity of intestinal stem cells: involvement of Wnt/ß-catenin signaling.

BACKGROUND: Early weaning and shorter breastfeeding duration are applied by a proportion of young mothers, especially in the social spheres of poverty-stricken areas. Early childhood is a critical period for intestinal development, which is driven by intestinal stem cells (ISCs). However, how early weaning practice affects the function of ISCs to mediate intestinal development remains unclear. METHODS: We established an excellent early weaning mice model that has significant intestinal atrophy and growth arrest symptoms to explore the responses of ISCs to early weaning. The primary and passaged intestinal organoids from the suckling or early weaning mice were cultured to explore the underlying mechanism of early weaning affecting the ISCs. RESULTS: Early weaning depressed the self-renewal of ISCs and attenuated the activity of ISCs-driven intestinal epithelial regeneration and crypt expansion in vivo and ex-vivo. Further results showed that early weaning retarded the differentiation of ISCs into transit-amplifying cells and Paneth cells, and accelerated the apoptosis of villous epithelial cells, jointly leading to intestinal epithelial atrophy. Mechanistically, early weaning inhibited Wnt signaling in ISCs, while an exogenous Wnt amplifier restored ISCs' function in ex-vivo. CONCLUSION: Our findings indicate that early weaning depresses the activity of ISCs via attenuating Wnt/ß-catenin signaling and triggers the proinflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-17 in jejunum, thereby impeding ISCs-driven epithelial regeneration and intestinal growth, which may provide a basal theory for the development of infant nutrients targeting stem cells to alleviate early weaning-induced intestinal problems.

Piwi maintains homeostasis in the Drosophila adult intestine.

PIWI genes are well known for their germline but not somatic functions. Here, we report the function of the Drosophila piwi gene in the adult gut, where intestinal stem cells (ISCs) produce enteroendocrine cells and enteroblasts that generate enterocytes. We show that piwi is expressed in ISCs and enteroblasts. Piwi deficiency reduced ISC number, compromised enteroblasts maintenance, and induced apoptosis in enterocytes, but did not affect ISC proliferation and its differentiation to enteroendocrine cells. In addition, deficiency of zygotic but not maternal piwi mildly de-silenced several retrotransposons in the adult gut. Importantly, either piwi mutations or piwi knockdown specifically in ISCs and enteroblasts shortened the Drosophila lifespan, indicating that intestinal piwi contributes to longevity. Finally, our mRNA sequencing data implied that Piwi may achieve its intestinal function by regulating diverse molecular processes involved in metabolism and oxidation-reduction reaction.

Elevation of Intracellular Alpha-Ketoglutarate Levels Inhibits Osteoclastogenesis by Suppressing the NF-κB Signaling Pathway in a PHD1-Dependent Manner.

Age-related osteoporosis, a high-prevalence disease in the aged population, is generally attributed to the excessive activity of osteoclasts. Most approved drugs treat osteoporosis by inhibition of osteoclasts. Although in vivo studies have shown that alpha-ketoglutarate (AKG), an intermediate in the TCA cycle, can ameliorate age-related osteoporosis, the effects of AKG on osteoclastogenesis and the underlying mechanism of its action have not been studied yet. Here, we showed that the elevation of intracellular AKG levels by supplementing dimethyl AKG (DM-AKG, a cell-permeable derivative of AKG) inhibits the receptor activator of NF-κB ligand (RANKL)-induced osteoclasts differentiation from primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells in vitro. We further found that DM-AKG treatment suppresses NF-κB signaling and oxidative phosphorylation (OXPHOS) during RANKL-induced osteoclastogenesis in RAW264.7 cells. Interestingly, dimethyl oxalylglycine (DMOG), an AKG competitive inhibitor of AKG-dependent prolyl hydroxylases (PHDs), antagonizes the suppression of the RANKL-activated NF-κB signaling pathway caused by DM-AKG treatment. Furthermore, blocked PHD1 expression (also known as EglN2), instead of PHD2 or PHD3, was confirmed to reverse the DM-AKG treatment-induced suppression of the RANKL-activated NF-κB signaling pathway. Accordingly, blocked PHD1 expression antagonized the inhibitory effects of DM-AKG on osteoclastogenesis. Together, our finding suggests that the elevation of intracellular AKG levels inhibits osteoclastogenesis by suppressing RANKL-activated NF-κB signaling in a PHD1-dependent manner, which may provide a novel nutritional strategy for osteoporosis treatment.

A comprehensive review on natural phenolic compounds as alternatives to in-feed antibiotics.

Intensive livestock and poultry farming in China largely relied on the use of in-feed antibiotics until July 2020. The consequences of antibiotic overuse in animal feed include accumulation in animal products and the development of bacterial antibiotic resistance, both of which threaten food safety and human health. China has now completely banned the circulation of commercial feed containing growth-promoting drug additives (except Chinese herbal medicine). Therefore, alternatives to in-feed antibiotics in animal production are greatly needed. Natural phenolic compounds (NPCs) exist widely in plants and are non-toxic, non-polluting, highly reproducible, and leave little residue. Many natural flavonoids, phenolic acids, lignans, and stilbenes have polyphenol chemical structures and exhibit great potential as alternatives to antibiotics. In this review we delineate the characteristics of plant-derived NPCs and summarize their current applications as alternatives to in-feed antibiotics, aiming to provide new strategies for antibiotic-free feeding and promote the development of more sustainable animal husbandry practices.

Stop codon readthrough alters the activity of a POU/Oct transcription factor during Drosophila development.

BACKGROUND: A number of cellular processes have evolved in metazoans that increase the proteome repertoire in relation to the genome, such as alternative splicing and translation recoding. Another such process, translational stop codon readthrough (SCR), generates C-terminally extended protein isoforms in many eukaryotes, including yeast, plants, insects, and humans. While comparative genome analyses have predicted the existence of programmed SCR in many species including humans, experimental proof of its functional consequences are scarce. RESULTS: We show that SCR of the Drosophila POU/Oct transcription factor Ventral veins lacking/Drifter (Vvl/Dfr) mRNA is prevalent in certain tissues in vivo, reaching a rate of 50% in the larval prothoracic gland. Phylogenetically, the C-terminal extension is conserved and harbors intrinsically disordered regions and amino acid stretches implied in transcriptional activation. Elimination of Vvl/Dfr translational readthrough by CRISPR/Cas9 mutagenesis changed the expression of a large number of downstream genes involved in processes such as chromatin regulation, neurogenesis, development, and immune response. As a proof-of-principle, we demonstrate that the C-terminal extension of Vvl/Dfr is necessary for correct timing of pupariation, by increasing the capacity to regulate its target genes. The extended Vvl/Dfr isoform acts in synergy with the transcription factor Molting defective (Mld) to increase the expression and biosynthesis of the steroid hormone ecdysone, thereby advancing pupariation. Consequently, late-stage larval development was prolonged and metamorphosis delayed in vvl/dfr readthrough mutants. CONCLUSIONS: We demonstrate that translational recoding of a POU/Oct transcription factor takes place in a highly tissue-specific and temporally controlled manner. This dynamic and regulated recoding is necessary for normal expression of a large number of genes involved in many cellular and developmental processes. Loss of Vvl/Dfr translational readthrough negatively affects steroid hormone biosynthesis and delays larval development and progression into metamorphosis. Thus, this study demonstrates how SCR of a transcription factor can act as a developmental switch in a spatiotemporal manner, feeding into the timing of developmental transitions between different life-cycle stages.

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila.

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.

Regulation of immune and tissue homeostasis by Drosophila POU factors.

The innate immune system of insects deploys both cellular and humoral reactions in immunocompetent tissues for protection of insects against a variety of infections, including bacteria, fungi, and viruses. Transcriptional regulation of genes encoding antimicrobial peptides (AMPs), cytokines, and other immune effectors plays a pivotal role in maintenance of immune homeostasis both prior to and after infections. The POU/Oct transcription factor family is a subclass of the homeodomain proteins present in all metazoans. POU factors are involved in regulation of development, metabolism and immunity. Their role in regulation of immune functions has recently become evident, and involves control of tissue-specific, constitutive expression of immune effectors in barrier epithelia as well as positive and negative control of immune responses in gut and fat body. In addition, they have been shown to affect the composition of gut microbiota and play a role in regulation of intestinal stem cell activities. In this review, we summarize the current knowledge of how POU transcription factors control Drosophila immune homeostasis in healthy and infected insects. The role of POU factor isoform specific regulation of stem cell activities in Drosophila and mammals is also discussed.

The POU/Oct Transcription Factor Nubbin Controls the Balance of Intestinal Stem Cell Maintenance and Differentiation by Isoform-Specific Regulation.

Drosophila POU/Oct transcription factors are required for many developmental processes, but their putative regulation of adult stem cell activity has not been investigated. Here, we show that Nubbin (Nub)/Pdm1, homologous to mammalian OCT1/POU2F1 and related to OCT4/POU5F1, is expressed in gut epithelium progenitor cells. We demonstrate that the nub-encoded protein isoforms, Nub-PB and Nub-PD, play opposite roles in the regulation of intestinal stem cell (ISC) maintenance and differentiation. Depletion of Nub-PB in progenitor cells increased ISC proliferation by derepression of escargot expression. Conversely, loss of Nub-PD reduced ISC proliferation, suggesting that this isoform is necessary for ISC maintenance, analogous to mammalian OCT4/POU5F1 functions. Furthermore, Nub-PB is required in enteroblasts to promote differentiation, and it acts as a tumor suppressor of Notch RNAi-driven hyperplasia. We suggest that a dynamic and well-tuned expression of Nub isoforms in progenitor cells is required for maintaining gut epithelium homeostasis.

Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis.

Gut immunity is regulated by intricate and dynamic mechanisms to ensure homeostasis despite a constantly changing microbial environment. Several regulatory factors have been described to participate in feedback responses to prevent aberrant immune activity. Little is, however, known about how transcriptional programs are directly tuned to efficiently adapt host gut tissues to the current microbiome. Here we show that the POU/Oct gene nubbin (nub) encodes two transcription factor isoforms, Nub-PB and Nub-PD, which antagonistically regulate immune gene expression in Drosophila. Global transcriptional profiling of adult flies overexpressing Nub-PB in immunocompetent tissues revealed that this form is a strong transcriptional activator of a large set of immune genes. Further genetic analyses showed that Nub-PB is sufficient to drive expression both independently and in conjunction with nuclear factor kappa B (NF-κB), JNK and JAK/STAT pathways. Similar overexpression of Nub-PD did, conversely, repress expression of the same targets. Strikingly, isoform co-overexpression normalized immune gene transcription, suggesting antagonistic activities. RNAi-mediated knockdown of individual nub transcripts in enterocytes confirmed antagonistic regulation by the two isoforms and that both are necessary for normal immune gene transcription in the midgut. Furthermore, enterocyte-specific Nub-PB expression levels had a strong impact on gut bacterial load as well as host lifespan. Overexpression of Nub-PB enhanced bacterial clearance of ingested Erwinia carotovora carotovora 15. Nevertheless, flies quickly succumbed to the infection, suggesting a deleterious immune response. In line with this, prolonged overexpression promoted a proinflammatory signature in the gut with induction of JNK and JAK/STAT pathways, increased apoptosis and stem cell proliferation. These findings highlight a novel regulatory mechanism of host-microbe interactions mediated by antagonistic transcription factor isoforms.

The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota.

BACKGROUND: Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood. RESULTS: We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism. CONCLUSIONS: This study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.