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Schematic figure of small intestinal villus and enterocytes: AA-ORS treatment increases rapidly dividing stem cells that are Lgr5 + , as well as proliferation markers p-Erk, p-Akt and PCNA. The treatment also increases cleaved caspase-3, p53 and Bcl-2. The AA-ORS treatment increases villus height, increased expression of NHE3, SGLT1 and β -galactosidases thereby increasing electrolyte absorption, sodium-coupled glucose absorption, and breakdown of disaccharides at the brush border membrane, respectively. A cartoon of the enterocyte on the top right shows the functional improvement in NHE3 mediated Na + absorption and glucose-coupled sodium transport with AA-ORS.
Source publication
Destruction of clonogenic cells in the crypt following irradiation are thought to cause altered gastrointestinal function. Previously, we found that an amino acid-based oral rehydration solution (AA-ORS) improved gastrointestinal function in irradiated mice. However, the exact mechanisms were unknown. Electrophysiology, immunohistochemistry, qPCR,...
Context in source publication
Context 1
... This study signifies how a system- atic selection of certain nutrients based on their beneficial effect on GI function helped to improve in situ intes- tinal stem cell proliferation, maturation, and differentiation, leading to functionally active long villus epithelial cells whose function and height were initially compromised by irradiation (Fig. 7). The study also supports Brain J. Leibowitz et al. 's observation that bone marrow derived stem cells have no significant role in the repopulation of intestinal mucosa following high dose radiation 14 . Future studies should seek to determine the mechanisms by which these amino acids increase the stem cell population, increase their ...
Citations
... Recently, we have identified that specific amino acid permeases (membrane proteins involved in the transport of amino acids into cells) played important roles in determining Cryptococcus neoformans' radiation sensitivity (2). Furthermore, a series of studies reported that treatment with a specific amino acid-based oral rehydration solution (AA-ORS) decreased intestinal paracellular permeability and plasma endotoxin, increased electrolyte absorption, and improved intestinal mucosal barrier function in male Swiss mice exposed to total body irradiation (TBI) (3)(4)(5). Oral drug administration represents a favorable option for the development of radiation countermeasures, particularly in the context of radiation casualties. Furthermore, the ingredients in the AA-ORS are all listed as "Generally Recognized as Safe" by the US FDA, exempting them from standard Federal Food, Drug, and Cosmetic Act (FFDCA) requirements. ...
... Consequently, there is an urgent demand for new MCMs capable of effectively mitigating and treating multiple organ injuries induced by radiation in diverse populations following a radiological or nuclear mass casualty incident (10). Recently, Gupta et al. (3,4) reported that treatment with AA-ORS could decrease radiation-induced GI injury, increase electrolyte absorption, prevent antigen translocation, and reduce inflammation, subsequently improving intestinal mucosal barrier function in male NIH Swiss mice exposed to TBI. Amino acids are fundamental components present in cells, acting as the basic elements of mammalian cellular machinery and serving as the building blocks for protein and enzyme synthesis. ...
To date, few FDA-approved medical countermeasures are available for addressing hematopoietic acute radiation syndrome (H-ARS). In this study, we present our latest research findings focusing on the evaluation of a novel radiation mitigator known as the mitigating amino acid mixture (MAAM). MAAM is composed of five amino acids as the recently reported amino acid-based oral rehydration solution for mitigating gastrointestinal (GI)-ARS. CD2F1 male and female mice were exposed to ⁶⁰Co-γ total body irradiation (TBI) at 9.0 or 9.5 Gy. Following irradiation, mice were orally administered with MAAM or a saline vehicle control once daily for a duration of 14 days, commencing 24 h after TBI. Mouse survival and body weight change were monitored for 30 days after irradiation. Complete blood counts (CBCs), bone marrow (BM) stem and progenitor cell survival (clonogenicity), and a serum cytokine antibody array were analyzed using samples from day 30 surviving mice. Our data revealed that MAAM treatment significantly enhanced survival rates in irradiated male CD2F1 mice, and the survival rate increased from 25% in the vehicle control group to 60% in the MAAM-treated group (p < 0.05) after 9.0 Gy TBI. The number of BM colonies significantly increased from 41.8 ± 6.4 /10⁴ cells (in the vehicle group) to 78.5 ± 17.0 /10⁴ cells (in the MAAM group) following 9.0 Gy TBI. Furthermore, MAAM treatment led to a decrease in the levels of six cytokines/proteins [cluster of differentiation 40 (CD40), interleukin (IL)-17A, C–X–C motif chemokine 10 (CXCL10/CRG-2), cutaneous T cell-attracting chemokine (CTACK), macrophage inflammatory protein (MIP)-3β, and IL-1β] and an increase in the levels of five other cytokines/proteins [IL-3Rβ, IL-5, leptin, IL-6, and stem cell factor (SCF)] in mouse serum compared to the vehicle group after 9.0 Gy TBI. However, similar alleviating effects of MAAM were not observed in the irradiated CD2F1 female mice. The serum cytokine profile in the irradiated female mice was different compared to the irradiated male mice. In summary, our data suggest that the beneficial effects of the mitigative amino acid combination treatment after radiation exposure may depend on sex.
... Aromatic amino acids, including TRP, PHE, and TYR, were reported to attenuate intestinal inflammation through the activation of calcium-sensing receptors in pigs [166]. Furthermore, mice exposed to radiation and treated with an amino acid-based oral rehydration solution (a mixture of threonine, valine, serine, tyrosine, and tryptophan) displayed enhanced intestinal epithelial proliferation with decreased paracellular permeability [167]. TRP alike PHE is an essential amino acid; yet, it has low tissue storage, and its overall concentration in the body is the lowest among all amino acids. ...
In the first part of this article, the role of intestinal epithelial tight junctions (TJs), together with gastrointestinal dopaminergic and renin–angiotensin systems, are narratively reviewed to provide sufficient background. In the second part, the current experimental data on the interplay between gastrointestinal (GI) dopaminergic and renin–angiotensin systems in the regulation of intestinal epithelial permeability are reviewed in a systematic manner using the PRISMA methodology. Experimental data confirmed the copresence of DOPA decarboxylase (DDC) and angiotensin converting enzyme 2 (ACE2) in human and rodent enterocytes. The intestinal barrier structure and integrity can be altered by angiotensin (1-7) and dopamine (DA). Both renin–angiotensin and dopaminergic systems influence intestinal Na+/K+-ATPase activity, thus maintaining electrolyte and nutritional homeostasis. The colocalization of B0AT1 and ACE2 indicates the direct role of the renin–angiotensin system in amino acid absorption. Yet, more studies are needed to thoroughly define the structural and functional interaction between TJ-associated proteins and GI renin–angiotensin and dopaminergic systems.
... The study hypothesis was based on previous observations that glucose stimulates calcium-activated chloride secretion in small intestine cells and that amino acid-based ORS may enhance intestinal epithelial proliferation and mucosal integrity, promoting nutrient absorption and intestinal health. 17,28 This study is among the first randomized controlled trials in children to compare a non-glucose amino acidbased alternative ORS formulation to the low osmolarity WHO-ORS formula. In this randomized controlled trial of young children with acute non-cholera watery diarrhoea, VS002A proved to be safe and effective in correcting ongoing fluid and electrolyte loss. ...
Background
Diarrhoeal disease poses a significant global health challenge, especially in children under three years old. Despite the effectiveness of oral rehydration therapy (ORT), its adoption remains low. Glucose-based ORS (GORS) is the standard, but novel formulations like glucose-free amino acid-based VS002A have emerged as potential alternatives. This study aimed to compare the safety and efficacy of VS002A against the standard WHO-ORS in treating non-cholera acute watery diarrhoea in children.
Methods
A triple-blind, randomized trial enrolled 310 male infants and children aged 6–36 months, who were assigned to receive WHO-ORS or VS002A over a 16-month period, from June 2021 to September 2022. Both groups received standard of care, including zinc supplementation. The Primary study outcome measured was the duration of diarrhoea. Secondary outcomes included stool output, treatment failure and adverse events. Exploratory endpoints included urinary output, body weight changes, blood biochemistry, stool microbiology and gut health biomarkers.
Findings
Both VS002A and WHO-ORS were well-tolerated with a low adverse event rate. While not different statistically (p = 0.10), duration of diarrhoea was shorter in children treated with VS002A vs. WHO-ORS (65.4 h vs. 72.6 h). Similarly, stool output was also lower vs. WHO-ORS in children treated with VS002A, though not statistically different (p = 0.40). Serum citrulline levels, an indicator of gut health, were higher in the VS002A group at 24 h suggesting a potential protective effect (p = 0.06).
Interpretation
The findings of this study support the non-inferiority of VS002A, a glucose-free amino acid-based ORS compared to the WHO-ORS standard of care. VS002A was shown to be safe and effective in treating non-cholera acute watery diarrhoea in young children. VS002A may offer advantages in pathogen-driven diarrhoea, supported by trends toward a lower duration of diarrhoea and stool output within the per protocol group. Furthermore, individuals with prolonged diarrhoea, severe malnutrition, environmental enteric dysfunction or have issues with obesity or insulin resistance, could benefit from a glucose-free ORS. This research contributes to addressing the persistent challenge of childhood diarrhoea by presenting an alternative glucose-free ORS formulation with potential advantages in select scenarios, offering a promising avenue for improving paediatric diarrhoea management worldwide.
Funding
The study was funded by Entrinsic Bioscience, LLC., Norwood, MA, USA.
... Evidence of these effects include decreased expression of transmembrane claudin-2 protein, increased occludin proteins and e-cadherin proteins, improved crypt and villus morphology, and LGR5+ stem cell proliferation. 17,[19][20][21] The effect of a tailored amino acid beverage on intestinal epithelial status and systemic responses; however, was not demonstrated within human participants performing EHS until recently. 12 Whereby, in response to EHS and compared with a water control, two amino acid beverage blends [i.e., 4.5 g/L (valine, aspartic acid, serine, threonine, and tyrosine) and 6.4 g/L (aspartic acid, serine, valine, isoleucine, threonine, and tyrosine)] presented attenuating effects on intestinal epithelial cell injury, luminal pathogenic translocation, and systemic inflammatory responses, without exacerbating gastrointestinal symptoms commonly associated with the exercise-induced gastrointestinal syndrome (EIGS) pathophysiological pathway. ...
... The acute intake of a novel commercial amino acid formulation (4.5 g/L: valine, aspartic acid, serine, threonine, and tyrosine) for up to 6-day supplementation period has been demonstrated to protect and restore murine intestinal epithelial barrier function in response to total body irradiation (Gupta et al., 2020;Yin et al., 2016) and severe exertional-heat stress (King et al., 2019). The formulation resulted in histological evidence of protected intestinal epithelium in association with reduced permeability, decreases in transmembrane claudin-2 protein expression, increases in occludin and e-cadherin proteins, improvements in crypt and villus morphology, and LGR5+ stem cell proliferation (Choi et al., 2017;Gupta et al., 2020;Yin et al., 2014Yin et al., , 2016. ...
... The acute intake of a novel commercial amino acid formulation (4.5 g/L: valine, aspartic acid, serine, threonine, and tyrosine) for up to 6-day supplementation period has been demonstrated to protect and restore murine intestinal epithelial barrier function in response to total body irradiation (Gupta et al., 2020;Yin et al., 2016) and severe exertional-heat stress (King et al., 2019). The formulation resulted in histological evidence of protected intestinal epithelium in association with reduced permeability, decreases in transmembrane claudin-2 protein expression, increases in occludin and e-cadherin proteins, improvements in crypt and villus morphology, and LGR5+ stem cell proliferation (Choi et al., 2017;Gupta et al., 2020;Yin et al., 2014Yin et al., , 2016. Another noncommercial amino acid formulation (6.4 g/L: aspartic acid, serine, valine, isoleucine, threonine, and tyrosine), designed to optimize intestinal fluid delivery (Funnell et al., 2023), may also offer protective functions (dual functionality) based on several key amino acid ratios but requires confirmation. ...
... It is speculated that the beneficial effect seen with amino acid beverage intervention was likely due to: (a) amino acid provisions improved cellular stability of the enterocyte phospholipid bilayer against local hypoperfusion, oxidative, and/or inflammatory processes; and/or supported an enhanced epithelial homeostasis (i.e., rapid cell proliferation and differentiation), albeit rationale from animal and/or in vitro models (Xiao et al., 2020). Within these experimental models, it is acknowledged and recognized that combining amino acids, as per the current beverage intervention, may play a beneficial role within intestinal epithelial cell stability and/or function (Choi et al., 2017;Gupta et al., 2020;King et al., 2019;Yin et al., 2014Yin et al., , 2016; and subsequently influence membrane permeability, translocation of pathogenic agents, and systemic immune responses, and (b) the provisions of amino acids during exercise and contribution to luminal content may have maintained some form of villi microvascular perfusion (Matheson et al., 2000), as proposed with previous whole protein experimental trials in humans . From a formulation and translational perspective, considering two independent amino acid beverage interventions were tested on two independent randomly allocated cohort of participants and resulted in similar outcomes compared with a CON; it is convincing that an amino acid intervention has a protective effect on epithelial cells against potential internal or external destructive stimuli. ...
The study aimed to determine the effects of two differing amino acid beverage interventions on biomarkers of intestinal epithelial integrity and systemic inflammation in response to an exertional-heat stress challenge. One week after the initial assessment, participants ( n = 20) were randomly allocated to complete two exertional-heat stress trials, with at least 1 week washout. Trials included a water control trial (CON), and one of two possible amino acid beverage intervention trials (VS001 or VS006). On VS001 (4.5 g/L) and VS006 (6.4 g/L), participants were asked to consume two 237-ml prefabricated doses daily for 7 days before the exertional-heat stress, and one 237-ml dose immediately before, and every 20 min during 2-hr running at 60% maximal oxygen uptake in 35 °C ambient conditions. A water volume equivalent was provided on CON. Whole blood samples were collected pre-, immediately post-, 1 and 2 hr postexercise, and analyzed for plasma concentrations of cortisol, intestinal fatty acid protein, soluble CD14, and immunoglobulin M (IgM) by ELISA, and systemic inflammatory cytokines by multiplex. Preexercise resting biomarker concentrations for all variables did not significantly differ between trials ( p > .05). A lower response magnitude for intestinal fatty acid protein (mean [95% CI]: 249 [60, 437] pg/ml, 900 [464, 1,336] pg/ml), soluble CD14 (−93 [−458, 272] ng/ml, 12 [−174, 197] ng/ml), and IgM (−6.5 [−23.0, 9.9] MMU/ml, −10.4 [−16.2, 4.7] MMU/ml) were observed on VS001 and V006 compared with CON ( p < .05), respectively. Systemic inflammatory response profile was lower on VS001, but not VS006, versus CON ( p < .05). Total gastrointestinal symptoms did not significantly differ between trials. Amino acid beverages’ consumption (i.e., 4.5–6.4 g/L), twice daily for 7 days, immediately before, and during exertional-heat stress ameliorated intestinal epithelial integrity and systemic inflammatory perturbations associated with exercising in the heat, but without exacerbating gastrointestinal symptoms.
... Mucosal height was measured by the same investigator as the mean vertical distance between the muscularis mucosa and the lumen surface at the villus tip ( Figure 3B). Villus height was the mean vertical distance between the base of each villus and the villus tip calculated from 20 villi in two different mice per group which were randomly selected (Yin et al., 2016). The same villi were used to calculate the mean width measured as a horizontal distance between the lateral surface epithelia at the middle of each villus. ...
... In this study, the increased conductance and FITC-dextran permeation were observed mostly in the jejunum and not in the ileum, suggesting that EHS-induced mucosal injury being confined mostly to the leaky regions of the small intestine such as the jejunum. A large body of evidence implies that circulating markers and indicators of the innate immune response (i.e., cytokines and chemokines) are directly related to increases in gut permeability and subsequently the severity of injury in critical illness (Casey et al., 1993;Clark & Coopersmith, 2007;Yin et al., 2016). Here, we show differences in gut permeability as measured by changes in conductance (Figure 2A,B) and FITC-dextran permeation ( Figure 2C,D) between treatment groups at 30 min recovery without changes in the innate immune response or markers of organ damage, suggesting that widespread systemic inflammation is not specifically derived from the gut following EHS. ...
Increased gut permeability is implicated in the initiation and extent of the cytokine inflammatory response associated with exertional heat stroke (EHS). The primary objective of this study was to determine if a five amino acid oral rehydration solution (5AAS), specifically designed for the protection of the gastrointestinal lining, would prolong time to EHS, maintain gut function and dampen the systemic inflammatory response (SIR) measured during EHS recovery. Male C57/BL6J mice instrumented with radiotelemetry were gavaged with 150 μL of 5AAS or H2 O, and ≈12 h later were either exposed to an EHS protocol where mice exercised in a 37.5°C environmental chamber to a self-limiting maximum core temperature (Tc,max) or performed the exercise control (EXC) protocol (25°C). 5AAS pretreatment attenuated hypothermia depth and length (p < 0.005), which are indicators of EHS severity during recovery, without any effect on physical performance or thermoregulatory responses in the heat as determined by percent body weight lost (≈9%), max speed (≈6 m/min), distance (≈700 m), time to Tc,max (≈160 min), thermal area (≈550°C∙min), and Tc,max (42.2°C). EHS groups treated with 5AAS showed a significant decrease in gut transepithelial conductance, decreased paracellular permeability, increased villus height, increased electrolyte absorption and changes in tight junction protein expression pattern suggestive of improved barrier integrity (p < 0.05). No differences were witnessed between EHS groups in acute phase response markers of liver, circulating SIR markers, or indicators of organ damage during recovery. These results suggest that a 5AAS improves Tc regulation during EHS recovery through maintaining mucosal function and integrity.
... A number of these stressors are known to result in the inhibition of translation, particularly calorie and nutrient deprivation 61,62 . Indeed, it has been shown that amino acid levels can have a significant effect on ISCs, although studies have focused on amino acid supplementation rather than depletion, and have been very limited in scope 63,64 . ...
The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5-expressing intestinal stem cells (ISCs). However, several Lgr5-negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5-negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity.
... Preclinical studies have also shown that Enterade can reverse radiation-induced injury that leads to a breakdown in GI structure (Gupta et al., 2020), increase weight gain and provide a survival benefit in mice (Yin et al., 2014). It has also been studied with funding from the National Space Biomedical Research Institute in experiments that found that the drink given as a daily gavage improved electrolyte balance and GI absorption of nutrients in irradiated animals (Yin et al., 2016). ...
Over the past 20+ years, the U.S. Government has made significant strides in establishing research funding and initiating a portfolio consisting of subject matter experts on radiation-induced biological effects in normal tissues. Research supported by the National Cancer Institute (NCI) provided much of the early findings on identifying cellular pathways involved in radiation injuries, due to the need to push the boundaries to kill tumor cells while minimizing damage to intervening normal tissues. By protecting normal tissue surrounding the tumors, physicians can deliver a higher radiation dose to tumors and reduce adverse effects related to the treatment. Initially relying on this critical NCI research, the National Institute of Allergy and Infectious Diseases (NIAID), first tasked with developing radiation medical countermeasures in 2004, has provided bridge funding to move basic research toward advanced development and translation. The goal of the NIAID program is to fund approaches that can one day be employed to protect civilian populations during a radiological or nuclear incident. In addition, with the reality of long-term space flights and the possibility of radiation exposures to both acute, high-intensity, and chronic lower-dose levels, the National Aeronautics and Space Administration (NASA) has identified requirements to discover and develop radioprotectors and mitigators to protect their astronauts during space missions. In sustained partnership with sister agencies, these three organizations must continue to leverage funding and findings in their overlapping research areas to accelerate biomarker identification and product development to help safeguard these different and yet undeniably similar human populations – cancer patients, public citizens, and astronauts.
... Recently, it was noted that certain amino acids (AAs) can increase transporter abundance on the brush-border membrane (BBM) of enterocytes, and thus enhance nutrient absorption 7,8 . ...
... Here, the 4AAs failed to increase iron flux in duodenal epithelial sheets isolated from mice lacking intestinal DMT1; thus, stimulation of iron transport via the 4AA formulation probably did not involve chelation. Another plausible mechanism by which AAs may influence iron absorption is via transporter trafficking to the enterocyte BBM, a phenomenon that has been documented by us previously 7,8,12 . Supporting this possibility, one hour after peroral administration of the 4AA formulation to live mice, DMT1 protein levels increased in duodenal BBMVs. ...
Iron-deficiency anemia is common worldwide and typically treated by oral iron supplementation. Excess enteral iron, however, may cause pathological outcomes. Developing new repletion approaches is thus warranted. Previous experimentation revealed that select amino acids (AAs) induce trafficking of transporters onto the enterocyte brush-border membrane (BBM), and enhance electrolyte absorption/secretion. Here, we hypothesized that certain AA would increase abundance of the main intestinal iron importer, divalent metal-ion transporter 1 (DMT1), on the BBM of duodenal enterocytes, thus stimulating iron absorption. Accordingly, all 20 AAs were screened using an ex vivo duodenal loop / DMT1 western blotting approach. Four AAs (Asp, Gln, Glu, Gly) were selected for further experimentation, and combined into a new formulation. The 4AAs stimulated 59Fe transport in mouse duodenal epithelial sheets in Ussing chambers (⁓4-fold; p<0.05). In iron-deprived mice, oral intragastric administration of the 4AA formulation increased DMT1 protein abundance on the enterocyte BBM (by ~1.5-fold; p<0.05). The 4AAs also enhanced in vivo 59Fe absorption (by ⁓2-fold; p<0.05), even when ~26 µg of cold iron was included in the transport solution (equal to a human dose of ~73 mg). Additional experimentation suggested that intestinal DMT1 was required for enhancement of iron transport by the 4AAs. Select AA thus enhance iron absorption by inducing DMT1 trafficking onto the apical membrane of duodenal enterocytes. We speculate that further refinement of this new 4AA formulation will ultimately allow iron repletion at lower effective doses (thus mitigating negative side effects of excess enteral iron).
... EXOs and EXO@INS were stained with phosphotungstic acid (2%) for 3 min before observation. The gel electrophoresis and Coomassie staining experiments to detect the protein composition were performed as previously reported 16 ...
... After 10 min at 4 C, the cells were collected after centrifugation (12,000 rpm  10 min). Then, 3% glutaraldehyde was added and cells were stored at 4 C. Cells were post-fixed in osmium tetroxide (1%), dehydrated in graded acetone and embedded in araldite as previously described 16 . The grids were examined using an electron microscope (JEM-1400PLUS, Japan) at 80 kV. ...
... Thus, we investigated the mechanisms in terms of signaling pathways related to EXOs trafficking. Mitogen-activated protein kinases (MAPKs) include ERK1/2 and p38 coordinate responses and serve as key sensors of the energy status within a cell 16 . MAPK activation is reportedly beneficial for nutrient absorption 45 . ...
As endogenous courier vesicles, exosomes play crucial roles in macromolecule transmission and intercellular communication. Therefore, exosomes have drawn increasing attention as biomimetic drug-delivery vehicles over the past few years. However, few studies have investigated the encapsulation of peptide/protein drugs into exosomes for oral administration. Additionally, the mechanisms underlying their biomimetic properties as oral delivery vehicles remain unknown. Herein, insulin-loaded milk-derived exosomes ([email protected]) were fabricated and the in vivo hypoglycemic effect was investigated on type I diabetic rats. Surprisingly, [email protected] (50 and 30 IU/kg) elicited a more superior and more sustained hypoglycemic effect compared with that obtained with subcutaneously injected insulin. Further mechanism studies indicated that the origin of excellent oral-performance of milk-derived exosomes combined active multi-targeting uptake, pH adaptation during gastrointestinal transit, nutrient assimilation related ERK1/2 and p38 MAPK signal pathway activation and intestinal mucus penetration. This study provides the first demonstration that multifunctional milk-derived exosomes offer solutions to many of the challenges arising from oral drug delivery and thus provide new insights into developing naturally-equipped nanovehicles for oral drug administration.
