In vitro and in vivo digestibility of prebiotic galactooligosacharides synthesized by ß-galactosidase from Lactobacillus delbruecki subsp. bulgaricus CRL450.

BACKGROUND: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by ß-galactosidase (ß-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-ß-gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-ß-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general ß (1→6) linked products showed higher resistance compared to ß (1→3) oligosaccharides. CONCLUSION: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility.

Aged males disproportionately succumb to increased COVID-19 severity, hospitalization, and mortality compared to females. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) facilitate SARS-CoV-2 viral entry and may have sexually dimorphic regulation. As viral load dictates disease severity, we investigated the expression, protein levels, and activity of ACE2 and TMPRSS2. Our data reveal that aged males have elevated ACE2 in both mice and humans across organs. We report the first comparative study comprehensively investigating the impact of sex and age in murine and human levels of ACE2 and TMPRSS2, to begin to elucidate the sex bias in COVID-19 severity.

Generation of Caco-2 cells stably expressing CYP3A4·POR·UGT1A1 and CYP3A4·POR·UGT1A1*6 using a PITCh system.

The small intestine plays a critical role in the absorption and metabolism of orally administered drugs. Therefore, a model capable of evaluating drug absorption and metabolism in the small intestine would be useful for drug discovery. Patients with genotype UGT1A1*6 (exon 1, 211G > A) treated with the antineoplastic drug SN-38 have been reported to exhibit decreased glucuronide conjugation and increased incidence of intestinal toxicity and its severe side effects, including severe diarrhea. To ensure the safety of drugs, we must develop a drug metabolism and toxicity evaluation model which considers UGT1A1*6. In this study, we generated CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells for pharmaceutical research using a PITCh system. The CYP3A4·POR·UGT1A1 KI-Caco-2 cells were shown to express functional CYP3A4 and UGT1A1. The CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells were sensitive to SN-38-induced intestinal toxicity. We thus succeeded in generating CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells, which can be used in pharmaceutical research. We also developed an intestinal epithelial cell model of patients with UGT1A1*6 and showed that it was useful as a tool for drug discovery.

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases.

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Differences in Hydrolase Activities in the Liver and Small Intestine between Marmosets and Humans.

For drug development, species differences in drug-metabolism reactions present obstacles for predicting pharmacokinetics in humans. We characterized the species differences in hydrolases among humans and mice, rats, dogs, and cynomolgus monkeys. In this study, to expand the series of such studies, we attempted to characterize marmoset hydrolases. We measured hydrolase activities for 24 compounds using marmoset liver and intestinal microsomes, as well as recombinant marmoset carboxylesterase (CES) 1, CES2, and arylacetamide deacetylase (AADAC). The contributions of CES1, CES2, and AADAC to hydrolysis in marmoset liver microsomes were estimated by correcting the activities by using the ratios of hydrolase protein levels in the liver microsomes and those in recombinant systems. For six out of eight human CES1 substrates, the activities in marmoset liver microsomes were lower than those in human liver microsomes. For two human CES2 substrates and three out of seven human AADAC substrates, the activities in marmoset liver microsomes were higher than those in human liver microsomes. Notably, among the three rifamycins, only rifabutin was hydrolyzed by marmoset tissue microsomes and recombinant AADAC. The activities for all substrates in marmoset intestinal microsomes tended to be lower than those in liver microsomes, which suggests that the first-pass effects of the CES and AADAC substrates are due to hepatic hydrolysis. In most cases, the sums of the values of the contributions of CES1, CES2, and AADAC were below 100%, which indicated the involvement of other hydrolases in marmosets. In conclusion, we clarified the substrate preferences of hydrolases in marmosets. SIGNIFICANCE STATEMENT: This study confirmed that there are large differences in hydrolase activities between humans and marmosets by characterizing marmoset hydrolase activities for compounds that are substrates of human CES1, CES2, or arylacetamide deacetylase. The data obtained in this study may be useful for considering whether marmosets are appropriate for examining the pharmacokinetics and efficacies of new chemical entities in preclinical studies.

Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

CYP3A deficiency alters bile acid homeostasis and leads to changes in hepatic susceptibility in rats.

Cytochrome P450 3A (CYP3A) as an important enzyme metabolizes many drugs and a variety of endogenous substances. Bile acids (BA) regulate physiological function by activating BA receptors. In this study, CYP3A1/2 gene knockout (KO) and wild-type (WT) rats were used to investigate the regulatory effects of CYP3A on BA homeostasis and liver function. Compared with WT rats, BA concentrations in serum, liver and small intestine of CYP3A1/2 KO rats increased significantly, which was due to the decrease of catabolism and the increase of synthesis. In particular, the composition of serum BA (overall hydrophobicity) presented an age- and CYP3A-dependent manner. With the aging of WT rats, the serum BA became more hydrophobic, while this trend was delayed in CYP3A1/2 KO rats. Moreover, the level of serum total cholesterol, the precursor of BA synthesis, decreased by about 20% in CYP3A1/2 KO rats, which is due to the low synthesis but high biotransformation rate. The increase of BA pool further led to the change of transcription level of BA receptor in liver (pregnane X receptor) and small intestine (Takeda G-protein receptor 5), and affected the function and morphology of CYP3A1/2 KO rat liver. In conclusion, CYP3A is a key regulator of BA homeostasis in rats, especially in regulating BA pool size, composition and balance of anabolism, and prevents susceptibility to hepatotoxicity under BA overload.

Bone Marrow-Derived Cells Restore Functional Integrity of the Gut Epithelial and Vascular Barriers in a Model of Diabetes and ACE2 Deficiency.

RATIONALE: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. OBJECTIVE: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. METHODS AND RESULTS: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2-/y, Akita (type 1 diabetes mellitus), and ACE2-/y-Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2-/y-Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2-/y-Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. CONCLUSIONS: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2-/y-Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

Pre-weaning adaptation responses in piglets fed milk replacer with gradually increasing amounts of wheat.

Hyperprolific sows rear more piglets than they have teats, and to accommodate this, milk replacers are often offered as a supplement. Milk replacers are based on bovine milk, yet components of vegetable origin are often added. This may reduce growth, but could also accelerate maturational changes. Therefore, we investigated the effect of feeding piglets a milk replacer with gradually increasing levels of wheat flour on growth, gut enzyme activity and immune function compared with a diet based entirely on bovine milk. The hypothesis tested was that adding a starch component (wheat flour) induces maturation of the mucosa as measured by higher digestive activity and improved integrity and immunity of the small intestines (SI). To test this hypothesis, piglets were removed from the sow at day 3 and fed either a pure milk replacer diet (MILK) or from day 11 a milk replacer diet with increasing levels of wheat (WHEAT). The WHEAT piglets had an increased enzyme activity of maltase and sucrase in the proximal part of the SI compared with the MILK group. There were no differences in gut morphology, histopathology and gene expression between the groups. In conclusion, the pigs given a milk replacer with added wheat displayed immunological and gut mucosal enzyme maturational changes, indicatory of adaptation towards a vegetable-based diet. This was not associated with any clinical complications, and future studies are needed to show whether this could improve responses in the subsequent weaning process.

Improving the digestibility of cereal fractions of wheat, maize, and rice by a carbohydrase complex rich in xylanases and arabinofuranosidases: an in vitro digestion study.

BACKGROUND: Cereal co-products rich in dietary fibres are increasingly used in animal feed. The high fibre content decreases the digestibility and reduces the nutrient and energy availability, resulting in lower nutritive value. Therefore, this study investigated the ability of two carbohydrase complexes to solubilize cell-wall polysaccharides, in particular arabinoxylan (AX), from different cereal fractions of wheat, maize, and rice using an in vitro digestion model of the pig gastric and small intestinal digestive system. The first complex (NSPase 1) was rich in cell-wall-degrading enzymes, whereas the second complex (NSPase 2) was additionally enriched with xylanases and arabinofuranosidases. The extent of solubilization of insoluble cell-wall polysaccharides after in vitro digestion was evaluated with gas-liquid chromatography and an enzymatic fingerprint of the AX oligosaccharides was obtained with high-performance anion-exchange chromatography with pulsed amperometric detection. RESULTS: The addition of carbohydrase increased the digestibility of dry matter and solubilized AX in particular, with the greatest effect in wheat fractions and less effect in maize and rice fractions. The solubilization of AX (expressed as xylose release) ranged from 6% to 41%, and there was an increased effect when enriching with xylanases and arabinofuranosidases in wheat aleurone and bran of 19% and 14% respectively. The enzymatic fingerprint of AX oligosaccharides revealed several non-final hydrolysis products of the enzymes applied, indicating that the hydrolysis of AX was not completed during in vitro digestion. CONCLUSION: These results indicate that the addition of a carbohydrase complex can introduce structural alterations under in vitro digestion conditions, and that enrichment with additional xylanases and arabinofuranosidases can boost this effect in wheat, maize, and rice. © 2020 Society of Chemical Industry.

Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice.

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

Regional Proteomic Quantification of Clinically Relevant Non-Cytochrome P450 Enzymes along the Human Small Intestine.

Current challenges in accurately predicting intestinal metabolism arise from the complex nature of the intestine, leading to limited applicability of available in vitro tools as well as knowledge deficits in intestinal physiology, including enzyme abundance. In particular, information on regional enzyme abundance along the small intestine is lacking, especially for non-cytochrome P450 enzymes such as carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs). We used cryopreserved human intestinal mucosa samples from nine donors as an in vitro surrogate model for the small intestine and performed liquid chromatography tandem mass spectrometry-based quantitative proteomics for 17 non-cytochrome P450 enzymes using stable isotope-labeled peptides. Relative protein quantification was done by normalization with enterocyte marker proteins, i.e., villin-1, sucrase isomaltase, and fatty acid binding protein 2, and absolute protein quantification is reported as picomoles per milligram of protein. Activity assays in glucuronidations and sequential metabolisms were conducted to validate the proteomics findings. Relative or absolute quantifications are reported for CES1, CES2, five UGTs, and four SULTs along the small intestine: duodenum, jejunum, and ileum for six donors and in 10 segments along the entire small intestine (A-J) for three donors. Relative quantification using marker proteins may be beneficial in further controlling for technical variabilities. Absolute quantification data will allow for scaling factor generation and in vivo extrapolation of intestinal clearance using physiologically based pharmacokinetic modeling. SIGNIFICANCE STATEMENT: Current knowledge gaps exist in intestinal protein abundance of non-cytochrome P450 enzymes. Here, we employ quantitative proteomics to measure non-cytochrome P450 enzymes along the human small intestine in nine donors using cryopreserved human intestinal mucosa samples. Absolute and relative abundances reported here will allow better scaling of intestinal clearance.

Cell-Type-Specific, Ketohexokinase-Dependent Induction by Fructose of Lipogenic Gene Expression in Mouse Small Intestine.

BACKGROUND: High intakes of fructose are associated with metabolic diseases, including hypertriglyceridemia and intestinal tumor growth. Although small intestinal epithelia consist of many different cell types, express lipogenic genes, and convert dietary fructose to fatty acids, there is no information on the identity of the cell type(s) mediating this conversion and on the effects of fructose on lipogenic gene expression. OBJECTIVES: We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and apolipoprotein synthesis, that regulation depends on the fructose transporter solute carrier family 2 member a5 [Slc2a5 (glucose transporter 5)] and on ketohexokinase (Khk), and that regulation occurs only in enterocytes. METHODS: We compared lipogenic gene expression among different organs from wild-type adult male C57BL mice consuming a standard vivarium nonpurified diet. We then gavaged twice daily for 2.5 d fructose or glucose solutions (15%, 0.3 mL per mouse) into wild-type, Slc2a5-knockout (KO), and Khk-KO mice with free access to the nonpurified diet and determined expression of representative lipogenic genes. Finally, from mice fed the nonpurified diet, we made organoids highly enriched in enterocyte, goblet, Paneth, or stem cells and then incubated them overnight in 10 mM fructose or glucose. RESULTS: Most lipogenic genes were significantly expressed in the intestine relative to the kidney, liver, lung, and skeletal muscle. In vivo expression of Srebf1, Acaca, Fasn, Scd1, Dgat1, Gk, Apoa4, and Apob mRNA and of Scd1 protein increased (P < 0.05) by 3- to 20-fold in wild-type, but not in Slc2a5-KO and Khk-KO, mice gavaged with fructose. In vitro, Slc2a5- and Khk-dependent, fructose-induced increases, which ranged from 1.5- to 4-fold (P < 0.05), in mRNA concentrations of all these genes were observed only in organoids enriched in enterocytes. CONCLUSIONS: Fructose specifically stimulates expression of mouse small intestinal genes for lipid and apolipoprotein synthesis. Secretory and stem cells seem incapable of transport- and metabolism-dependent lipogenesis, occurring only in absorptive enterocytes.

Characterization of Differential Tissue Abundance of Major Non-CYP Enzymes in Human.

The availability of assays that predict the contribution of cytochrome P450 (CYP) metabolism allows for the design of new chemical entities (NCEs) with minimal oxidative metabolism. These NCEs are often substrates of non-CYP drug-metabolizing enzymes (DMEs), such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), carboxylesterases (CESs), and aldehyde oxidase (AO). Nearly 30% of clinically approved drugs are metabolized by non-CYP enzymes. However, knowledge about the differential hepatic versus extrahepatic abundance of non-CYP DMEs is limited. In this study, we detected and quantified the protein abundance of eighteen non-CYP DMEs (AO, CES1 and 2, ten UGTs, and five SULTs) across five different human tissues. AO was most abundantly expressed in the liver and to a lesser extent in the kidney; however, it was not detected in the intestine, heart, or lung. CESs were ubiquitously expressed with CES1 being predominant in the liver, while CES2 was enriched in the small intestine. Consistent with the literature, UGT1A4, UGT2B4, and UGT2B15 demonstrated liver-specific expression, whereas UGT1A10 expression was specific to the intestine. UGT1A1 and UGT1A3 were expressed in both the liver and intestine; UGT1A9 was expressed in the liver and kidney; and UGT2B17 levels were significantly higher in the intestine than in the liver. All five SULTs were detected in the liver and intestine, and SULT1A1 and 1A3 were detected in the lung. Kidney abundance was the most variable among the studied tissues, and overall, high interindividual variability (>15-fold) was observed for UGT2B17, CES2 (intestine), SULT1A1 (liver), UGT1A9, UGT2B7, and CES1 (kidney). These differential tissue abundance data can be integrated into physiologically based pharmacokinetic (PBPK) models for the prediction of non-CYP drug metabolism and toxicity in hepatic and extrahepatic tissues.

Comparison of CYP Induction by Coleus forskohlii Extract and Recovery in the Small Intestine and Liver of Mice.

We examined CYP induction and recovery at various doses of Coleus forskohlii extract (CFE) to assess potential drug interactions by a mechanism involving intestinal CYP. Mice were administered diets with various doses of CFE up to 0.5% (equivalent to 700-800 mg/kg body weight) for 2 weeks, then CFE was withdrawn for 3 d. Changes in CYP activities and mRNA expression in the small intestine and liver were then evaluated. CFE induced CYP in the small intestine at a higher dose compared to the liver; CYP3A was induced at 0.5% and 0.005% CFE in the small intestine and liver, respectively. There was no sex difference in CFE dose for CYP induction. CYP induction quickly reverted after withdrawal of CFE, especially for CYP3A, in the small intestine; whereas, a gradual recovery was observed in the liver. In conclusion, CFE induced CYP in the small intestine and liver; however, a higher dose of CFE was needed for the small intestine. Moreover, the induction was soon recovered, suggesting actual interactions of CFE with prescription drugs are unlikely to occur through CYP in the small intestine.

Whole pearl millet feeding does not impair performance and nutrient digestibility in 28-day-old broiler chickens.

The effects of varying inclusion levels of whole grain millet in millet-soya bean-based diets on growth performance, gizzard development, digesta characteristics and nutrient digestion in broiler chicken were investigated. Starter (0-14 days) and grower (15-28 days) broiler chicken diets containing pearl millet at 500 and 540 g/kg diet, respectively, were formulated. The diets comprised of 0%, 20%, 40%, 60%, 80% and 100% of millet incorporated as whole grain. One-day-old unsexed Arbor Acres Plus chicks (n = 540) were allotted to the experimental diets in a completely randomized design with the diets and water provided ad libitum for 28 days. Each treatment was replicated seven times, and each replicate had 12 chicks. Results showed that daily live weight gain and feed conversion ratio of chickens on the whole millet grain diets compared favourably with chicken on the control in both starter and grower phases, while feed intake reduced quadratically (p < .05) with increased whole grain millet levels in the starter phase. Morphological and structural characteristics of the gizzard and small intestine and intestinal digesta pH and viscosity were also unaffected (p > .05) by whole grain millet inclusion levels. However, the weight of intact millet grain in gizzard increased linearly (p < .001) with whole grain millet inclusion in the diets. Dietary whole grain millet inclusion also consistently lowered (p < .05) jejunal and ileal maltase and sucrase activities, but did not influence (p > .05) pancreatic amylase activity. Ileal crude protein and starch digestibility increased, while ileal energy digestibility decreased significantly (p < .05) with whole grain millet inclusion. Whole grain millet inclusion in broiler starter and grower diets up to 500-540 g/kg did not negatively impact on broiler chicken performance.

Activity and mRNA expression levels of selected cytochromes P450 in various sections of the human small intestine.

AIMS: To characterize mRNA expression levels (17 cytochromes P450) and activity (9 isoforms) of major cytochromes P450 expressed throughout the human small intestine. METHODS: Tissue samples were obtained from 9 deceased subjects and intestinal sections (n = 10) were isolated for each subject. Relative mRNA expression levels were determined using quantitative real-time PCR. Intestinal microsomes were prepared from 5 subsections: duodenum, jejunum (proximal and mid-jejunum) and ileum (proximal and mid-ileum) regions. In vitro incubations were performed with various cytochrome P450 probe substrates: bupropion (CYP2B6), repaglinide (CYP2C8), tolbutamide (CYP2C9), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), chlorzoxazone (CYP2E1), ebastine (CYP2J2), midazolam (CYP3A4/5) and lauric acid (CYP4A11). Metabolite formation was assessed using validated liquid chromatography-tandem mass spectrometry assays. RESULTS: Cytochrome P450 mRNA levels ranked as follows: CYP3A4 > CYP2C9 > CYP2C19 > CYP2J2 > CYP4F2. Cytochrome P450 mRNA transcripts showed different patterns in their relative expression from 1 region to the other but CYP3A4, CYP2C9, CYP2C19 and CYP2J2 displayed the highest levels of mRNA expression (>5%) in all intestinal sections. Cytochrome P450 activities were greater in proximal part of the small intestine with the jejunum showing the greatest drug-metabolism activity. Spearman's correlation analyses indicated that cytochrome P450 mRNA expressions and corresponding cytochrome P450 activities in the human intestine were moderately associated for CYP2C19, CYP2D6 and CYP4A11 (rs  = 0.44-0.56). CONCLUSIONS: Our study provides new and additional information on the expression and activities of selected cytochromes P450 in various sections of the human small intestine.

Bioactivity-based analysis and chemical characterization of hypoglycemic and antioxidant components from Artemisia argyi.

Diabetes is one of the metabolic disorders in the world. It is the prime reason of mortality and morbidity owing to hyperglycemia which is link with numerus obstacles. Artemisia argyi is commonly used as an ingredient in healthy foods as well as an herbal medicine in Asian countries. The present research aims to evaluate the hypoglycemic effects of A. argyi and reveal its the potentially active constituents. The chemical composition was identified by HPLC-DAD-Q-TOF-MS, and fractionation was performed by extraction. The fractions were assessed by the blood glucose level, oral glucose tolerance and small intestinal α-glucosidase inhibitory tests, and an analysis of the total phenolic content (TPC), antioxidant and α-glucosidase inhibitory activities. In our efforts to characterize the compounds responsible for hypoglycemic effect, bioactivity-guided fraction of the MeOH extract and chemical investigation of its active EtOAc fraction led to the successful identification of caffeoylquinic acids, which were elucidated by molecular docking, using the crystal structure of S. cerevisiae isomaltase (PD code: 3AXI). In summary, this bio-guided search revealed that caffeoylquinic acids from A. argyi as potential active constituents displayed with hypoglycemic activity, which provided a basis for further study of pharmacological activity.

Species-Specific Glucose-6-Phosphatase Activity in the Small Intestine-Studies in Three Different Mammalian Models.

Besides the liver, which has always been considered the major source of endogenous glucose production in all post-absorptive situations, kidneys and intestines can also produce glucose in blood, particularly during fasting and under protein feeding. However, observations gained in different experimental animals have given ambiguous results concerning the presence of the glucose-6-phosphatase system in the small intestine. The aim of this study was to better define the species-related differences of this putative gluconeogenic organ in glucose homeostasis. The components of the glucose-6-phosphatase system (i.e., glucose-6-phosphate transporter and glucose-6-phosphatase itself) were analyzed in homogenates or microsomal fractions prepared from the small intestine mucosae and liver of rats, guinea pigs, and humans. Protein and mRNA levels, as well as glucose-6-phosphatase activities, were detected. The results showed that the glucose-6-phosphatase system is poorly represented in the small intestine of rats; on the other hand, significant expressions of glucose-6-phosphate transporter and of the glucose-6-phosphatase were found in the small intestine of guinea pigs and homo sapiens. The activity of the recently described fructose-6-phosphate transporter-intraluminal hexose isomerase pathway was also present in intestinal microsomes from these two species. The results demonstrate that the gluconeogenic role of the small intestine is highly species-specific and presumably dependent on feeding behavior (e.g., fructose consumption) and the actual state of metabolism.