bluuebunny/crossref_metadata_2025 huggingface dataset:
Nutrient‐Induced Metabolism Dictates Intestinal Epithelial Crypt Proliferation
Authors: Zhou Weinan , Clara Rosmarie , Mansouri Abdelhak , Langhans Wolfgang , Dailey Megan J.
Published: June 2021

The intestinal epithelium plays an essential role in nutrient absorption, hormone release and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make‐up of the tissue. We have found that glucose induces an increase in the rate of proliferation in intestinal epithelial crypts in a concentration dependent manner. The mechanism underlying this nutrient‐driven change in crypt proliferation is not known, but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to the increase in crypt proliferation. To test this hypothesis, we utilized primary small intestinal epithelial crypts from C57BL/6J mice to study the effect of glucose on crypt metabolism using a Seahorse XFe96 Extracellular Flux Analyzer for real‐time metabolic measurements. Glycolysis was determined by measuring the extracellular acidification rate (ECAR) in response to glucose and the glycolytic pathway inhibitor 2‐Deoxy‐D‐glucose (2‐DG). Glucose oxidation was determined by measuring oxygen consumption rate (OCR) in response to glucose and the glucose oxidation pathway inhibitor UK‐5099. Glucose‐induced mitochondrial respiration was determined by measuring OCR in response to glucose and modulators of mitochondrial respiration (i.e. ATP synthase inhibitor oligomycin, uncoupler FCCP, complex I inhibitor rotenone, complex III inhibitor antimycin A). The maximum respiration capacity (i.e. the capacity to oxidize any substrates present to meet metabolic challenges) was determined by measuring FCCP‐stimulated OCR. We found that glucose increased ECAR in a concentration dependent manner, and the glycolytic pathway inhibitor 2‐DG reduced glucose‐induced ECAR, indicating that glucose stimulates glycolysis of crypts. However, neither glucose nor the glucose oxidation pathway inhibitor UK‐5099 affected OCR, indicating that glucose oxidation is not active in crypts. Furthermore, glucose increased FCCP‐stimulated OCR, indicating that the presence of glucose is necessary for crypts to utilize substrates for oxidation. These data suggest that high nutrient availability drives metabolism towards utilization of glucose for glycolysis in crypts, and glucose might indirectly induce an increase in the oxidation of other nutrients (e.g. fatty acids, amino acids) in crypts. These metabolic changes may contribute to the nutrient‐induced crypt proliferation.

Support or Funding Information

USDA ILLU‐538‐926
Psychoactive Drugs as a Route to Development of Novel Anti‐parasitic Agents
Authors: Woodhouse Kristen , Chan John D , Marchant Jonathan
Published: June 2021

Over a third of the world's population is infected with parasitic worms. One of the most burdensome infections underpins the neglected tropical disease schistosomiasis (Bilharzia) caused by parasitic flatworms of the genus Schistosoma, which afflicts ~200 million people worldwide. Consequently, there is a need to discover and develop next generation anthelmintics, active against a broad spectrum of parasitic helminths.

Flatworm musculature is regulated by bioaminergic signalling: addition of exogenous 5‐HT to isolated flatworm muscle fibres causes contraction. This effect is likely mediated by engagement of serotonergic G protein coupled receptors (GPCRs). For example, in free living planarians, knockdown of a serotonergic GPCR (S7.1) impairs worm motility. Here, we demonstrate that the psychoactive agent, lysergic acid diethylamide (LSD) acts as a agonist at the planarian S7.1 receptor (EC50 = 1.3±0.5nM, Emax 99±5% of 5‐HT response). LSD evoked contraction inhibited the motility of free living planarian worms (distance moved 16±2% versus control worms) and potently blocked bipolar regeneration evoked by praziquantel (IC50 = 0.5±0.2nM). These data raises the possibility that other psychoactive drugs, including psychotropics with known activity at human 5‐HT2 receptors, could serve as efficacious lead compounds to disrupt flatworm mobility.

Therefore, we screened a variety of psychoactive agents on the motility of free living planarian flatworms, as well as the functionality of heterologously expressed S7.1 using a real time cAMP biosensor. Agents were discovered that modulated flatworm movement and regeneration, and efficacy of the screened molecules provided information about structural features necessary for activity at this abundant flatworm serotonergic GPCR. These data also identify features of ligands conveying activity at flatworm 5‐HT GPCRs.

Support or Funding Information

Supported by NSF (MCB1615538, to JSM).
Prostaglandins Stimulate cAMP Generation via Serosal Membrane Receptors in Porcine Epithelial Cells
Authors: Riley Melissa , Schultz Bruce
Published: June 2021

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a gene that codes for an anion channel that is regulated by cyclic adenosine monophosphate (cAMP) and its associated kinase, protein kinase A (PKA). Numerous physiological agonists are known to stimulate the activity of adenylyl cyclase, which generates cAMP. Our previous work demonstrated that acute exposure to prostaglandin (PG) D2 or PGE2 causes a rapid and sustained increase in short circuit current (Isc), an indicator of anion secretion, across cultured pig vas deferens epithelial cells, an outcome that is consistent with PG‐induced increases in adenylyl cyclase activity. Also, it was shown that indomethacin, an agent that blocks prostaglandin synthesis, precluded the effects of the stimulant bradykinin, which suggests that all components of the prostaglandin synthesis and the response pathways are present in these cells. However, the molecular identity of the prostaglandin receptor(s), the location(s) of these receptors and second messengers that mediate the responses remain to be determined. Thus, experiments were carried out to determine to localize receptors of PGD2 and PGE2 to either the mucosal or serosal aspect of the cells. These experiments were carried out with cultured primary adult pig vas deferens epithelial (1°PVD) cells using a modified Ussing‐style flux chamber. Serosal, but not mucosal exposure of PGE2 produced a rapid increase in Isc, suggesting serosal localization of EP receptors. Similarly, PGD2 and a selective DP receptor agonist, BW 245C, produced an effect following serosal exposure. Experiments were also carried out to determine the effect of PGs on cAMP generation using a cAMP enzyme immunoassay kit with 1°PVD cells. PGE2, PGD2, and BW 245C all showed an increase in stimulation of cAMP generation. This stimulation was inhibited by the presence of DP receptor antagonist, BW A868C, and EP2/EP4 receptor antagonists, PF‐04418948 and L‐161,982. The simplest interpretation of data is that both EP and DP receptors are localized to the serosal membrane of vas deferens epithelial cells and cAMP generation is affected by inhibition of these receptors. Activation of these receptors is tightly linked to anion secretion, which would be expected to increase pH and fluid volume in the male reproductive duct.

Support or Funding Information

IOS‐1238831
Inward currents through somatic Na/K‐ATPase α1‐subunit mutants are not required for hyperaldosteronism
Authors: Meyer Dylan , Gatto Craig , Artigas Pablo
Published: June 2021

Hyperaldosteronism (HA), the most common cause of secondary hypertension, is often initiated by an aldosterone‐producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations to several genes were identified in these adenomas, including to the Na/K‐ATPase (NKA) α1‐subunit. The membrane potential (Vm) of cells harboring these NKA‐α1 mutations is depolarized, which is believed to underlie the pathophysiological aspects of HA.

At physiological Na+ and K+ concentrations, wild type NKA activity generates outward current due to the export of 3 Na+ and import of 2 K+ per cycle against their gradients. Under similar conditions, all HA‐associated NKA mutants evaluated thus far have dramatically reduced activity and an inward current, mediated by Na+ or H+, proposed to be a ‘gain‐of‐function’ required for Vm depolarization. The outward current of wild type NKA typically contributes minimally to Vm. Thus, for the inward current through HA‐associated NKA mutants to significantly depolarize Vm, its amplitude is expected to be significantly larger than the amplitude of wild type NKA outward current. Assuming similar expression levels, the transport rate of inward current through NKA mutant pumps should be larger than the pumping turnover rate of a single wild type NKA.

To calculate the turnover rate of inward current through human NKA mutants, we utilized two‐electrode voltage clamp (TEVC) of Xenopus oocytes heterologously expressing HA‐associated mutants to measure the ouabain‐sensitive inward current amplitude in the presence of Na+, and 3H‐ouabain binding assays on the same oocyte. As ouabain binds to NKA at a 1:1 ratio, its quantity is equivalent to the number of pumps in the oocyte's plasma membrane. The ratio of current at −50 mV (a physiologically‐relevant voltage) to the moles of ouabain bound to the oocyte yielded inward current turnover rates of 22.0 ± 1.8 s−1 for L104R, 417.3 ± 29.4 s−1 for delF100‐L104, 60.7 ± 5.8 s−1 for V332G, and 19.1 ± 1.4 s−1 for EETA963S, while turnover rate of wild type K+‐induced pump current was 32.0 ± 1.5 s−1. This indicates that the inward current of several NKA mutants may not suffice to significantly depolarize Vm, and that the pathophysiological effects may simply reflect lack of function. Furthermore, oocytes expressing G99R, a mutant recently associated with HA, lacked inward current under physiological conditions, instead presenting K+‐induced outward pump current. TEVC measurements of extracellular K+‐induced current in G99R revealed a ~3‐fold reduction of K+ apparent affinity compared to wild type, while measurements of intracellular Na+‐dependent, ATP‐activated currents in giant inside‐out patches showed a ~3‐fold reduction of Na+ apparent affinity, demonstrating impaired function under physiological conditions. These results suggest that lack of function is the main mechanism leading to HA in all mutants.

Support or Funding Information

Supported by NSF MCB‐1515434
Moderate resistance exercise improves the metabolic profile of adipose tissue in a model of disuse
Authors: Baer Lisa A. , Harris Johan , Sindeldecker Devin , Wade Charles E. , Stanford Kristin I.
Published: June 2021

Treatment of trauma and several other maladies requires patient immobilization and restriction of physical activity, which can result in impaired metabolic health. In contrast to physical immobilization, physical activity and exercise improves metabolic health. Using a rodent model of disuse (hindlimb unloading) combined with resistance exercise, we determined if moderate resistance exercise could negate the effects of disuse on the metabolic profile of white adipose tissue (WAT). Male rats were assigned to four groups 1) Ambulatory No Exercise (ANE); 2) Ambulatory Exercise (AE); 3) Hindlimb Unloaded No Exercise (HUNE); 4) Hindlimb Unloaded Exercise (HUE). All rats completed a 10‐day pre‐training resistance exercise regiment prior to random group assignments. HUNE and HUE animals were placed in a tail traction system with their hindlimbs unloaded and were followed for 14 days. Animals designated to AE or HUE groups exercised for 14 days. At day 14, plasma and tissue samples were collected and analyzed. Resistance exercise resulted in a decrease in body mass in the ambulatory animals (AE vs. ANE), and hindlimb unloading significantly decreased body mass compared to ambulatory animals (HUNE and HUE vs. ANE and AE), but there was no additive effect of exercise on body mass (HUNE vs. HUE). Hindlimb unloading reduced hindlimb muscle mass (ANE and AE vs. HUNE and HUE), and resistance exercise partially restored soleus mass in the hindlimb unloaded animals (HUNE vs. HUE). Total white adipose tissue mass (WAT), perigonadal WAT (pgWAT) mass, and subcutaneous WAT (scWAT) mass and adipocyte size were significantly reduced in response to both exercise and hindlimb unloading. Expression of genes and proteins in scWAT involved in fatty acid oxidation and glucose metabolism were significantly increased with exercise (ANE vs. AE), reduced with hindlimb unloading (ANE vs. HUNE), and restored with exercise in the hindlimb unloaded animals (HUNE vs. HUE). In conclusion, these data indicate that moderate resistance exercise improves the metabolic profile of both pgWAT and scWAT in both the ambulatory state and in an animal model of disuse, providing a novel therapeutic benefit for the bed rest patients.

Support or Funding Information

US Army MRMC CDMRP W81XWH‐13‐1‐0489
Physiological Sensitivity to Salt and Uninephrectomy
Authors: Clemmer John , Pruett W. Andrew , Hester Robert
Published: June 2021

Salt sensitivity, with or without concomitant hypertension, is associated with increased mortality. Reduced functional renal mass plays an important role in causing salt sensitive hypertension for many individuals but the mechanisms are unclear. Moreover, different derangements in human physiology can result in the same phenotype. Methodologies that could discover patterns in sensitivity to dietary change or interventions would be a revolutionary tool in personalizing medicine. We used HumMod, an integrative mathematical model of human physiology, to simulate population responses to changes in salt intake with or without reduced kidney mass (uninephrectomy). A heterogeneous population of 1000 virtual patients was created by randomly varying important physiological parameters. We examined potential physiological mechanisms responsible for the chronic blood pressure (BP) response to salt (8‐fold change in salt intake for 3 weeks) with full kidney mass and again with a single kidney in the same group of virtual patients. We also used topological data analysis (TDA), a new clustering algorithm tool, to separate patient subpopulations. In the entire population, BP increased with increased salt intake (14 ± 3 mmHg). This was associated with small changes in glomerular filtration rate (GFR) but significant increases in renal blood flow, total peripheral resistance, and distal tubular sodium reabsorption. Interestingly, TDA distinguished 5 unique clusters of salt sensitive individuals (> 15 mmHg change in BP with increased salt). While these clusters had similar BP responses to salt, they were significantly different in the mechanisms of salt sensitivity (e.g. greater reductions in GFR or impairments in the renin angiotensin system.) After kidney removal in these same individuals, the three most salt sensitive clusters were associated with a blunted increase in renal blood flow (RBF), exaggerated increases in loop and distal sodium reabsorption, and greater sodium and volume retention as compared to the salt resistant population. These data suggest that the suppression of sodium reabsorption and renin angiotensin system is key for salt resistance, while RBF in addition to GFR may be an important factor when considering criteria for potential kidney donors.

Support or Funding Information

Supported by NSF EPS‐0903787, P20 GM104357, and NIH PO1 HL51971.
Comparison of Compensatory Reserve Index Before and After Analgesia
Authors: Hinojosa‐Laborde Carmen , Fernandez Jessie Renee D. , Muniz Gary W. , Nawn Corinne D. , Burns Rebecca K. , Le Thuan H. , Porter Kathy B. , Hardy John T. , Convertino Victor A.
Published: June 2021

BACKGROUND

Prehospital assessment of cardiovascular stability during hemorrhage is unpredictable because standard vital signs do not predict cardiovascular shock until it is too late for the medic to respond with a life‐saving intervention. In an effort to improve survival from hemorrhage, a machine learning algorithm has been developed as an early predictor of cardiovascular shock. The algorithm is able to detect reductions in central blood volume during blood loss, and assess the individual's capacity to compensate for blood loss by calculating beat‐ to‐beat compensatory reserve index (CRI) from a finger photoplethysmography waveform. CRI=100 represents normovolemia and CRI=0 represents hypovolemia at the point of cardiovascular decompensation. The algorithm has been validated in blood loss studies in animals and humans; however these studies were conducted in the absence of other confounding factors associated with blood loss such as trauma and pain. The current study was designed to determine if pain, in the absence of trauma, affects CRI by comparing CRI before and after epidural anesthesia to provide analgesia during childbirth. The purpose of this study was to test the hypothesis that pain or analgesia does not affect CRI.

METHODS

This study was conducted in healthy pregnant women (n=20) during the labor phase of childbirth. CRI was calculated from a finger pulse oximeter placed on the patient's index finger and connected to the DataOx™ CRI monitor. CRI was recorded for 1 hour before and 1 hour after epidural anesthesia.

RESULTS

CRI (mean ± SD) before analgesia was 83±11. CRI at 10, 20, 30, 40, 50, and 60 minutes after analgesia was 77±13, 80±13, 77±15, 81±16, 83±13, and 84±15, respectively. Repeated measures ANOVA revealed no statistical difference in CRI values before and after epidural anesthesia (p = 0.085). Pain scale scores before and after analgesia were 7±2 and 1±2, respectively.

CONCLUSIONS

These results indicate that pain, as an independent factor (in the absence of blood loss or trauma), does not affect compensatory reserve, and provide preliminary evidence that pain associated with trauma may not affect the body's ability to compensate for blood loss. This study supports the use of pain management on the battlefield, and supports the utility of assessing compensatory reserve in the prehospital setting to improve survivability from combat trauma.

Support or Funding Information

FUNDING: ­­­US Army MRMC Combat Casualty Care Research Program
GLP‐2 receptor in the brainstem is required for glucose homeostasis and insulin sensitivity
Authors: Shi Xuemei , Chacko Shaji , Brown Alexander , Li Feng , Guan Xinfu
Published: June 2021

Background and Objective

Glucagon‐like peptides (GLP‐1/2) are co‐secreted from enteroendocrine L cells in the gut and preproglucagon neurons in the brainstem, and are attributed through the gut‐brain axis to improving glucose homeostasis and insulin sensitivity after gastric bypass surgery. It is unknown if GLP‐2 receptor (GLP‐2R) in the brainstem plays a physiological role in glucose homeostasis. Our objective was to determine the physiological relevance of the brainstem GLP‐2R to glycemic control.

Methods and Results

1) Generation of a brainstem‐specific Glp2r knockout mouse model: First, we revealed that GLP‐2R protein was localized to Phox2b neurons in the brainstem dorsal vagal complex (DVC). Second, we generated Phox2b‐specific Glp2r knockout (PKO) mice by the Cre‐LoxP system, i.e., crossing Phox2b‐cre mice with Glp2r‐loxP‐flanked (floxed) mice. Third, we validated that Glp2r was deleted specifically in the DVC Phox2b neurons. 2) Characterization of the mouse metabolic phonotype: First, we showed that the PKO mice fed regular chow (~25 g BW) had higher blood glucose baseline after overnight fast, and lower blood glucose exclusion after ip glucose tolerance challenge. Second, we quantified glucose homeostasis and insulin sensitivity in conscious mice using hyperinsulinemic euglycemic clamp (coupled with dual stable isotopic tracers 6,6‐2H2‐d‐glucose and 2H2O). The PKO mice at the basal condition had higher endogenous glucose production (5.10 ± 0.25 vs 8.02 ± 0.26 mmol/kg/h, respectively, for the WT and PKO mice). During hyperinsulinemic euglycemic clamp, the PKO mice had lower (−16%) glucose infusion rate (GIR) as resulted mainly from a decrease (−15%) in insulin‐mediated stimulation of peripheral glucose utilization (Rd).

Conclusions

Mice with Glp2r deletion in the brainstem DVC Phox2b neurons displayed hyperglycemia, glucose intolerance and insulin resistance, suggesting that the brainstem GLP‐2R plays an important, physiological role in the control of glucose homeostasis and insulin sensitivity.

Support or Funding Information

(Supported by the USDA CRIS grant 3092‐5001‐059 and the National NSFC grant 81570722).
Specificity of Compensatory Reserve vs SMO2, Lactate and ETCO2 as Early Predictors of Tolerance to Progressive Reductions in Central Blood Volume
Authors: Convertino Victor A , Howard Jeff T , Schiller Alicia M
Published: June 2021

Circulatory shock resulting from acute uncontrolled hemorrhage remains a leading cause of death in both military and civilian trauma. Early, accurate and reliable prediction of decompensation is associated with effective intervention and improved clinical outcomes. Individual tolerance to reduced central blood volume can serve as a model to assess the sensitivity and specificity of vital sign measurements. The compensatory reserve (CR) is a measurement of this capacity. Measurements of muscle oxygen saturation (SmO2), lactate and end tidal CO2 (ETCO2) have recently gained attention as potentially superior prognostic tools for early assessment of the status of patients with progressive hemorrhage, but fail to identify individual tolerance. We hypothesized that CR would be able to better predict hemodynamic compensation and provide greater specificity and sensitivity than SmO2, lactate, or ETCO2. To test this hypothesis, we employed lower body negative pressure (LBNP) on healthy human subjects until symptoms of presyncope were evident. Receiver operating characteristic area under the curve (ROC AUC), sensitivity and specificity were used to evaluate the ability of CRI and SmO2, lactate and ETCO2 to predict hemodynamic decompensation. CR had the superior ability to predict decompensation compared to SM02, lactate and ETCO2 (ROC AUC: 0.91 vs. 0.68; 0.54; 0.70 p<0.0007), respectively. Similarly, CR also exhibited a greater sensitivity (0.87 vs. 0.65; .85, .89; p<0.05) and specificity (0.80 vs. 0.63, 0.29, 0.54, p<0.05). These findings support the notion that CR provides superior detection of hemodynamic compensation compared to measurements of tissue oxygenation, lactate and ETCO2.

Support or Funding Information

US Army MRMC Combat Casualty Care Research Program
Discovery and biosynthesis of hybrid polyketide‐nonribosomal peptides in nematodes
Authors: Butcher Rebecca A. , Feng Likui , Shou Qingyao
Published: June 2021

Polyketides and nonribosomal peptides are well‐known for their antibiotic, antifungal, immunosuppressant, anti‐parasitic, and anti‐cancer activities. These structurally complex natural products are biosynthesized by polyketide synthases (PKSs) and nonribosomal synthetases (NRPSs), modular megasynthases that function in either an assembly‐line or iterative manner. Although polyketides and nonribosomal peptides are produced by many species of bacteria, fungi, and plants, they are extremely rare in metazoans. The genome of the nematode Caenorhabditis elegans is predicted to encode a huge, multi‐module, hybrid PKS/NRPS (PKS‐1), as well as a multi‐module NRPS (NRPS‐1), with unknown functions. Using comparative metabolomics and NMR spectroscopy, we identified and elucidated the chemical structure of nemamide, a hybrid polyketide‐nonribosomal peptide that is biosynthesized by PKS‐1/NRPS‐1 using both iterative and assembly‐line mechanisms. In recent work, we used CRISPR‐Cas9 to make specific mutations in pks‐1 and nrps‐1 in order to investigate the roles of specific enzymatic domains in the biosynthesis of nemamide. We showed that nemamide promotes survival during starvation‐induced larval arrest and facilitates recovery from arrest in response to food. Nemamide is produced in the CAN neurons under starvation conditions and downregulates insulin/ insulin‐like growth factor‐1 (IGF‐1) signaling. Our results uncover a novel mechanism by which animals respond to nutrient fluctuations to extend survival. Homologs of PKS‐1 and NRPS‐1 are present in most nematode species, including parasitic ones, and thus, nemamide likely plays an evolutionarily conserved role across nematode species.

Support or Funding Information

We acknowledge the support of the NIH/NIGMS, NSF, and Ellison Medical Foundation.
Application of Metabolomics and Fluxomics to Study Fatty Acid Synthesis in Alternative Crops
Authors: Alonso Ana Paula , Tsogtbaatar Enkhtuul , Cocuron Jean‐Christophe
Published: June 2021

The goal of this research is to address the need for an alternative supply of jet fuel using native plants that do not compete with food crops. Pennycress (Thlaspi arvense), a member of the Brassicaceae, has been identified as a promising bioenergy crop: the chemical composition of its oil is ideally suited as a renewable source of biodiesel and jet fuel. This plant has tremendous potential for large‐scale oil production, has a short maturity time and is not a food crop. Pennycress can be commercially propagated as a winter annual, and has therefore the potential to be grown off‐season, complementing, rather than competing, the production of commodity crops, and serving as a winter cover crop. However, for this plant to become an economically viable source of specialty fuels, molecular and genetic resources need to be developed to facilitate and enhance oil production by breeding and/or genetic manipulation. This study aims to find potential biochemical step(s) that limit(s) oil synthesis, which will serve as targets for future crop improvement.

To advance towards this goal, we determined the intracellular metabolite levels (metabolomics) in pennycress embryos at different stages of development. For this purpose, we have developed novel and highly sensitive methodologies using state‐of‐the‐art liquid chromatography tandem mass spectrometry (LC‐MS/MS). This metabolomics study highlighted the metabolites and pathways that were active in pennycress embryos and important for oil production. The contribution of each pathway to fatty acid synthesis in terms of carbon, reductant and energy provision is being assessed by measuring the carbon flow through the metabolic network. We are hence performing a 13C‐Metabolic Flux Analysis (fluxomics) in developing pennycress embryos to build a map of carbon flow through central metabolism. This study describes the combination of innovative tools that will pave the way for controlling seed composition in promising alternative crops.

Support or Funding Information

J.C.C was supported in part by the Agriculture and Food Research Initiative competitive grant # 2016‐67013‐24605 of the USDA National Institute of Food and Agriculture. This study is supported by the USDA‐U.S. Department of Energy Plant Feedstocks in Genomics for Bioenergy under Award Number DE‐SC0016490.
The Bioavailability and Distribution of Bovine Milk Exosomes is Distinct from that of their Cargos in Mice
Authors: Manca Sonia , Giraud David , Zempleni Janos
Published: June 2021

Background

Among extracellular vesicles, exosomes are of particular interest because of their roles in cell‐to‐cell communication, which is facilitated by the delivery of cargos such as RNAs, to recipient cells. Dietary exosomes in bovine milk are transported by intestinal and vascular endothelial cells in human and rodent cell cultures, and delivered to peripheral tissues in mice. Little is known about the fate of the cargos in milk exosomes following absorption.

Hypothesis

RNAs in bovine milk exosomes are delivered to distinct peripheral tissues in mice.

Methods

Exosomes were isolated from bovine milk using ultracentrifugation. Exosome membranes were labeled using the fluorophore, DiR, whereas RNA cargos were labeled using EXO‐Glow Red in separate experiments. Identity, integrity, fine dispersion, and count of exosomes were assessed using transmission electron microscopy, western blots, and nanoparticle tracker (not shown). Exosomes were administered orally by gavage (1×1012 exosomes/g) in BALB/c mice. Absorption and distribution of exosomes and their cargos was monitored at timed intervals for up to 24 h using an iBox® Small Animal Imaging system and LiCor Odyssey CLx. At timed intervals, mice were euthanized and tissues were collected for densitometry analysis using VisionWorks®LS and Image Studio software.

Results

At t = 12 h after administration of DiR‐labeled exosomes by gavage, only a faint DiR signal was detected in peripheral tissues (Fig. 1); labeled RNA was detected in all peripheral tissues with a preference for kidneys, brain, lungs and liver. Figure 2 depicts the densitometry analysis of labeled RNA, corrected by the signal produced by unconjugated Exo‐glow. At t = 24 h, the majority of absorbed exosomes accumulated in liver (Fig. 3), whereas the RNA cargo accumulated preferably in brain and kidneys (Figs. 4,5).

Conclusions

Bovine milk exosomes and their RNA cargos are bioavailable and accumulate in distinct tissues in mice. Our data suggest that bovine milk exosomes are disassembled in the intestinal mucosa, and RNA cargos are repackaged in endogenous exosomes for transfer to tissues, preferably brain and kidneys.

Future studies

We will conduct a comprehensive analysis of milk whole exosomes and RNA cargo kinetics in mice to obtain an accurate assessment of bioavailability, distribution, and elimination phenomena in health and disease.

Support or Funding Information

Support: NIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.
Dried Plum's Unique Ability to Prevent and Reverse Bone Loss in Ovarian Hormone Deficiency: Efficacy and Possible Mechanisms
Authors: Arjmandi Bahram H. , Johnson Sarah A. , Pourafshar Shirin H. , Navaei Negin H. , George Kelli S. , Akhavan Neda S.
Published: June 2021

Osteoporosis is a chronic disorder associated with aging that is characterized by a loss of bone mass and quality leading to an increased risk of fragility fractures. Postmenopausal women are at the greatest risk of developing osteoporosis due to a cessation in ovarian hormone production, thus leading to accelerated bone loss. As the demographic shifts to a more aged population, a growing number of postmenopausal women will be afflicted with osteoporosis. A search for promising non‐pharmacological alternative therapies for osteoporosis is of prime importance as medications used for the prevention and treatment of osteoporosis can be associated with side effects and low compliance. Certain lifestyle factors, including nutrition, are known to reduce the risk of developing osteoporosis and therefore play an important role in bone health. In terms of nutrition, dried plum (prune) has been shown to be one of the most efficacious interventions for preventing and reversing bone loss in an ovariectomized rat model of osteoporosis as well as in postmenopausal women. The purpose of this presentation is to provide an overview of the efficacy of dried plum for preventing and reversing bone loss associated with ovarian hormone deficiency and to highlight possible mechanisms of action. We end with the results of a follow‐up study demonstrating that postmenopausal women taking dried plums during our one year clinical trial conducted five years earlier retained bone mineral density compared to the control. Overall, these findings suggest that dried plum is a promising functional food therapy for postmenopausal bone loss with the potential for a long‐lasting bone‐protective effect.

Support or Funding Information

USDA, California Dried Plum, Abbott Laboratories
The remarkable, recently extinct “mole‐duck” Talpanas lippa (Aves: Anseriformes) from Kauai, Hawaii: behavioral implications of its neuroanatomy and skull morphology
Authors: Witmer Lawrence M. , Ridgely Ryan C. , James Helen F. , Olson Storrs L. , Iwaniuk Andrew N.
Published: June 2021

Divergent evolution of island birds is well documented, and Talpanas lippa, from ~5400‐year‐old lake deposits in Kauai, may be among the strangest cases. Talpanas has synapomorphies of Anatidae and potentially oxyurines. Postcranial bones indicate that Talpanas was probably flightless and terrestrial. We microCT scanned the preserved skull elements followed by 3D visualization of the brain endocast and inner ear. We also microCTed heads of other ducks (Anas, Mergus, Oxyura), as well as those of potential analogs (kiwi, platypus), and generated brain cast, inner ear, eyeball, and skull models. The neuroanatomy of Talpanas was like that of no other known bird. The visual system was extraordinarily reduced, as evidenced by the small optic nerves and the almost indiscernible optic lobes, which, consistent with the small eyeball size as judged by orbital osteology, suggest that Talpanas was not very reliant on sight. Kiwis (Apteryx) also have very reduced visual systems, but not to the extent seen in Talpanas. Kiwis, on the other hand, have dramatically expanded the olfactory apparatus, but, in Talpanas, the olfactory bulbs were unexpanded and comparable to those of other anatids. Without question, the major sensory modality used by Talpanas was the somatosensory (touch) system, as shown by the unparalleled expansion of the trigeminal system. Kiwis and, to a lesser extent, oxyurines have enlarged trigeminal ganglia, but in Talpanas they were enormous, dominating the ventral aspect of the brain cast and excavating the skull internally and externally. Perhaps a better extant analog for Talpanas is the platypus, which likewise has reduced vision, moderate olfaction, and is a somatosensory specialist with an expanded trigeminal system. The palate and braincase indicate that Talpanas had a broad low bill that was richly supplied with tactile nerve endings. Muscle scars and mandibular differences may signal a feeding apparatus that also diverged from its dabbling duck heritage. The picture of Talpanas that emerges is of a very different duck: a flightless, weakly‐sighted, potentially nocturnal animal foraging with its broad sensate bill in the moist underbrush of Kauai.

Support or Funding Information

US National Science Foundation (IOB‐0517257, IOS‐1050154, IOS‐1456503 to LMW), and the Ohio University Heritage College of Osteopathic Medicine
Dietary Flavonoid Intake and Body Measures Related to CVD Risk: Results from What We Eat in America, NHANES 2007–2010
Authors: Sebastian Rhonda S , Enns Cecilia Wilkinson , Goldman Joseph D , Moshfegh Alanna J
Published: June 2021

Flavonoids appear to reduce risk of cardiovascular disease (CVD). Obesity, especially abdominal obesity, increases CVD risk. Whereas multiple studies have found inverse associations between flavonoid intake and either BMI or waist circumference (WC), no research has examined whether flavonoid intake is related to class of CVD risk as based on a combination of these variables. The purpose of this study was to investigate in a large, nationally representative sample of U.S. adults whether flavonoid intake is related to having combined body measures that put one at very or extremely high risk for diabetes, high blood pressure, and CVD (hereafter termed “very high CVD risk”). Data from one 24‐hour recall for 5,232 males and 5,306 non‐pregnant, non‐lactating females age ≥20 years participating in What We Eat in America (WWEIA), NHANES 2007–2010 were analyzed. CVD risk was assessed based on BMI and WC, as described in NIH National Heart, Lung, and Blood Institute guidelines. Very high CVD risk was defined as either BMI = 30.0–34.9 and large WC (>102 cm for men, >88 cm for women) or BMI ≥ 35.0 (any WC). Intakes of total flavonoids and five flavonoid classes (anthocyanidins, flavan‐3‐ols, flavanones, flavones, and flavonols) were determined using the recently released, comprehensive database Flavonoid Values for USDA Survey Foods and Beverages 2007–2010. For total flavonoids and for each class, individuals were divided into four categories of intake. Percentages of individuals at very high CVD risk by category of flavonoid intake were estimated using logistic regression with adjustment for confounding variables. Trends in the percentage at very high CVD risk were assessed via orthogonal polynomial contrasts. One‐third of adults (34%) were classified at very high CVD risk. For all adults, inverse trends were observed between percentages of individuals at very high CVD risk and intakes of anthocyanidins, flavan‐3‐ols, and flavanones (p<0.01). Having very high CVD risk was less likely among individuals in the highest versus those in the lowest intake category for anthocyanidins (OR: 0.65; 99% CI: 0.54, 0.79) and flavan‐3‐ols (OR: 0.68; 99% CI: 0.54, 0.85). Higher anthocyanidin intake was associated with a lower percentage of individuals at very high CVD risk among both men and women; for flavan‐3‐ols, this relationship was observed for men only, and for flavanones it was found for women only. No significant associations were seen between intakes of total flavonoids, flavones, or flavonols and very high CVD risk. Among U.S. adults, intakes of some classes of flavonoids, in particular those contributed primarily by tea and fruits (but not vegetables), are inversely associated with levels of BMI and WC that are predictive of high or extremely high CVD risk.

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ARS, USDA.
Metals and immunity
Authors: Nolan Elizabeth M.
Published: June 2021

Metal‐sequestering innate immune proteins participate in the host/pathogen interaction. The S100 protein family includes calprotectin (S100A8/S100A9), psoriasin (S100A7), and S100A12, and these proteins exhibit antimicrobial activity that results from their ability to chelate transition metals in the extracellular space and thereby reduce the availability of essential metal nutrients to pathogens. Calprotectin is a versatile metal‐sequestering protein that can capture a number of divalent first‐row transition metals at an unusual hexahistidine site, whereas psoriasin and S100A12 can selectively deplete zinc. Recent biochemical and biophysical studies that address the coordination chemistry and host‐defense function of these abundant human proteins will be presented.

Support or Funding Information

This research was supported by the NSF (CHE‐1352132), the Camille & Henry Dreyfus Foundation, and the MIT Center for Environmental Health Sciences (NIH Grant P30‐ES002109).
Vitamin K Content of U.S. Dairy Foods and the Association with Fat Content
Authors: Fu Xueyan , Harshman Stephanie G , Shen Xiaohua , Haytowitz David , Booth Sarah L
Published: June 2021

Vitamin K is an essential nutrient that has been implicated in cardiovascular disease and other chronic diseases. There are multiple forms of vitamin K. Phylloquinone (PK, vitamin K1) is plant‐based and has been well‐quantified in the US diet. Menaquinones (MK, vitamin K2) are a class of vitamin K compounds that differ from PK in the length and saturation of their side chain. Although MK are found in animal products and fermented food, their contents of foods have not been well characterized. The objectives of this study were to: 1) quantify, using mass spectrometry technology, the different forms of MK (MK4 through MK13 in the following commonly consumed dairy foods: milk (n=40 samples), yogurt (n=17), Greek yogurt (n=16), and cheeses (n=81); and 2) compare the MK contents of full‐fat, reduced‐fat and non‐fat milk, yogurt, Greek yogurt, cottage cheese and cheddar cheese. All dairy samples were either obtained through USDA's National Food and Nutrient Analysis Program or purchased from retail outlets.

Total vitamin K concentrations in full fat yogurt, Greek yogurt, cottage cheese and cheddar cheese were 28.1±18.9, 30.1±6.5, 54.2±8.3 and 28.2±41.3 μg/100 g, respectively. The total vitamin K content of blue cheese, fresh cheese, soft‐ripened cheese and semi‐soft cheese was 439±129, 309±204, 495±214 and 310±126 μg/100 g, respectively. Non‐fermented cheeses, like processed cheese, contained lower amounts of vitamin K (90.9±37.9 μg /100 g). Full fat dairy products contained appreciable amounts of MK, primarily in the forms of MK9, MK10 and MK11, which together accounted for 90% of total vitamin K in dairy foods. We also measured modest amounts of MK4 and MK12. In contrast, there was little MK5, MK6, MK7 or MK13 detected in the majority of dairy products.

Total vitamin K content of full fat milk, 2% milk, 1% milk and non‐fat milk was 38.1±8.6, 19.4±7.7, 12.9±2.0 and 7.7±2.9 μg/100 g, respectively. Reduced fat or fat free dairy products (Greek yogurt, yogurt, cottage cheese and cheddar cheese) contained 8–22% of the vitamin K found in full fat products.

To the best of our knowledge, this is the first report of MK contents of U.S. dairy products. The vitamin K contents are influenced by the fat content of the dairy products. If biological activity of MK from foods is established, dairy could have an important dietary role in vitamin K nutrition.

Support or Funding Information

Supported by the USDA, Agricultural Research Service under Cooperative Agreement No. 58‐1950‐7‐707 and No. 58‐1235‐1‐193, and Dairy Management Inc.
Metallothionein‐2A mRNA in Peripheral Blood Mononuclear Cells Reflects Dietary Zinc Intake in Healthy Adults
Authors: Hennigar Stephen R. , Karl J. Philip , Kelley Alyssa M. , Anderson Bradley J. , Armstrong Nicholes J. , McClung Holly L. , Berryman Claire E. , McClung James P.
Published: June 2021

Zinc is an essential nutrient for humans;however, a sensitive biomarker to assess zinc status has not been identified. Metallothionein(MT)‐1 and –2 are ubiquitously expressed proteins that bind cellular zinc with high affinity. MT expression is induced through binding of zinc to metal‐regulatory transcription factor 1, which then binds to metal‐responsive elements in the promoter of the metallothionein gene. The objective of this study was to determine whether expression of MT1 and/or MT2 in peripheral blood mononuclear cells (PBMCs) is associated with dietary zinc intake in healthy human adults. Forty‐three male and female volunteers (33.6 ± 14.3 y, BMI 25.6 ±2.86 kg/m2) completed a Block Food Frequency Questionnaire (v. 2014)to assess habitual dietary zinc intake. Blood was collected and plasma and PBMCs were isolated. RNA was isolated from PBMCs and expression of MT1A and MT2A mRNA was determined by RT‐qPCR. Plasma MT2A protein was determined by ELISA and plasma zinc concentration was determined by atomic absorption spectroscopy. Average daily zinc intake was 15.5 ± 7.44 mg/d (range: 4.89‐31.7mg/d). Plasma zinc concentration (10.8 ± 1.01 μmol/L) did not reflect dietaryzinc intake (r=0.0890; P=0.586). PBMC MT2Aexpression was 9‐fold greater than MT1A (11.5E‐3± 0.0127, 1.30E‐3 ± 0.00527; P<0.05). MT1Aexpression in PBMCs was not correlated with dietary zinc intake (r=0.137; P=0.383). MT2A expression in PBMCs was positively correlated with dietary zinc intake (r= 0.302; P<0.05) and increased with increasing levels of dietary zinc (<11 mg zinc/d, 7.29E–3 ± 0.00881; 11–20mg zinc/d, 9.93E–3 ± 0.0141; >20 mg zinc/d, 15.4E–3 ± 0.0114; P<0.05). Plasma MT2A protein was not correlated with dietary zinc intake (r=−0.0840;P=0.605). Findings suggest that MT2A mRNA in PBMCs reflects dietary zinc intake in healthy adults and may be a component in determining zinc status.

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The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. Support provided by USAMRMC.
Seven Day Blackberry Feeding Lowers the Respiratory Quotient in Males And Improves Insulin Sensitivity
Authors: Solverson Patrick , Rumpler William , Leger Jayme L , Redan Benjamin , Ferruzzi Mario G , Baer David J , Novotny Janet A
Published: June 2021

Berries and/or berry extracts have been shown to reduce adiposity in animal models consuming a high fat diet, possibly due to alterations in energy substrate utilization. The objective of this research was to test the effect of berry intake on energy substrate use in humans consuming a high fat diet. In a placebo controlled crossover study design with 2 treatment periods, overweight/obese men (n=27) were fed a high fat diet (40% en fat) containing either 600 g/d blackberries (BB, 1200 mg/d flavonoids) or 600 g/d sweetened gelatin (calorie matched, flavonoid‐free control) for 1 week prior to a meal‐based glucose tolerance test (MTT) (75 g of sugar from waffle and syrup) and 24h stay in a room‐sized indirect calorimeter. Across the 24 hr period, the average RQ was significantly lower with the BB treatment (0.810 vs. 0.817, BB vs. gelatin, p=0.040) while energy expenditure (EE) was not different (2485 vs. 2439 kcal, p=0.11, BB vs. gelatin treatments). During the MTT, AUC of glucose was not different between BB and gelatin treatment (3488 vs. 4070 mg*min/dL, respectively, p=0.12). However, AUC of insulin was lower after consumption of BB compared to gelatin (6485 vs. 8245 uU*min/ml, p=0.0002). During this 4 hr MTT period, EE was not statistically different between the 2 treatments (BB, 452 kcal; gelatin, 440 kcal, p=0.20). However, there was a significant reduction in the RQ with BBs (BB, 0.84; gelatin, 0.85, p=0.004) which was independent of age or BMI. During the 4 hr period that started with dinner, the comparisons were similar, where a reduction in RQ and an increase in fat oxidation were observed with the BB treatment (0.818 vs. 0.836, 28 vs. 24 g, respectively; BB vs. gelatin treatments) with no difference in EE (493 vs. 483, p=0.26, BB vs. gelatin). The 24 hr stay in the calorimeter included 30 min of treadmill walking in the afternoon following lunch. During this time period, again there was a significant reduction in RQ with BB treatment (0.856 vs 0.871; BB vs. gelatin). Blackberry consumption may contribute to increased fat oxidation and improved glucose sensitivity in overweight males on a high fat diet. This is the first study to demonstrate the potential for blackberries to combat the metabolic effects of a high fat diet in human subjects. Mechanistic studies aimed at describing biochemical changes leading to these observations are underway.

Support or Funding Information

Supported by the U.S. Department of Agriculture.