Intestinal absorption of D-fructose isomers, D-allulose, D-sorbose and D-tagatose, via glucose transporter type 5 (GLUT5) but not sodium-dependent glucose cotransporter 1 (SGLT1) in rats.

D-allulose, D-sorbose and D-tagatose are D-fructose isomers that are called rare sugars. These rare sugars have been studied intensively in terms of biological production and food application as well as physiological effects. There are limited papers with regard to the transporters mediating the intestinal absorption of these rare sugars. We examined whether these rare sugars are absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) as well as via GLUT type 5 (GLUT5) using rats. High-fructose diet fed rats, which express more intestinal GLUT5, exhibited significantly higher peripheral concentrations, Cmax and AUC0­180 min when D-allulose, D-sorbose and D-tagatose were orally administrated. KGA-2727, a selective SGLT1 inhibitor, did not affect the peripheral and portal vein concentrations and pharmacokinetic parameters of these rare sugars. The results suggest that D-allulose, D-sorbose and D-tagatose are likely transported via GLUT5 but not SGLT1 in rat small intestine.

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.

Bone Growth is Influenced by Fructose in Adolescent Male Mice Lacking Ketohexokinase (KHK).

Fructose is metabolized in the cytoplasm by the enzyme ketohexokinase (KHK), and excessive consumption may affect bone health. Previous work in calcium-restricted, growing mice demonstrated that fructose disrupted intestinal calcium transport. Thus, we hypothesized that the observed effects on bone were dependent on fructose metabolism and took advantage of a KHK knockout (KO) model to assess direct effects of high plasma fructose on the long bones of growing mice. Four groups (n = 12) of 4-week-old, male, C57Bl/6 background, congenic mice with intact KHK (wild-type, WT) or global knockout of both isoforms of KHK-A/C (KHK-KO), were fed 20% glucose (control diet) or fructose for 8 weeks. Dietary fructose increased by 40-fold plasma fructose in KHK-KO compared to the other three groups (p < 0.05). Obesity (no differences in epididymal fat or body weight) or altered insulin was not observed in either genotype. The femurs of KHK-KO mice with the highest levels of plasma fructose were shorter (2%). Surprisingly, despite the long-term blockade of KHK, fructose feeding resulted in greater bone mineral density, percent volume, and number of trabeculae as measured by µCT in the distal femur of KHK-KO. Moreover, higher plasma fructose concentrations correlated with greater trabecular bone volume, greater work-to-fracture in three-point bending of the femur mid-shaft, and greater plasma sclerostin. Since the metabolism of fructose is severely inhibited in the KHK-KO condition, our data suggest mechanism(s) that alter bone growth may be related to the plasma concentration of fructose.

Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types.

BACKGROUND: Mammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia. RESULTS: Here we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired. CONCLUSIONS: Differentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.

Antimicrobial Activity of the Phenolic Compounds of Prunus mume against Enterobacteria.

Mume fruit, the Japanese apricot (Prunus mume SIEB. et ZUCC.), is popular in Japan and is mostly consumed in the pickled form called umeboshi. This fruit is known to have anti-microbial properties, but the principal constituents responsible for the antimicrobial properties have not yet been elucidated. We investigated the antimicrobial activities of the phenolic compounds in P. mume against enterobacteria. In this study, growth inhibitory activities were measured as an index of the antibacterial activities. The phenolic compounds were prepared from a byproduct of umeboshi called umesu or umezu (often translated as "mume vinegar"). Umesu or umezu phenolics (UP) contain approximately 20% phenolic compounds with p-coumaric acid as a standard and do not contain citric acid. We observed the inhibitory effects of UP against the growth of some enterobacteria, at a relatively high concentration (1250-5000 µg/mL). Alkali hydrolysates of UP (AHUP) exhibited similar antibacterial activities, but at much lower concentrations of 37.5-300 µg/mL. Since AHUP comprises hydroxycinnamic acids such as caffeic acid, p-coumaric acid, and ferulic acid, the antibacterial activities of each of these acids were examined. Our study shows that the phenolic compounds in P. mume other than citric acid contribute to its antimicrobial activity against enterobacteria in the digestive tract.

Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells.

Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and goblet-enriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing.NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.

Safety and adherence of Umezu polyphenols in the Japanese plum (Prunus mume) in a 12-week double-blind randomized placebo-controlled pilot trial to evaluate antihypertensive effects.

OBJECTIVES: Medications or lifestyle changes to prevent or improve hypertension often press considerable efforts on patients suffering from mild hypertension. Capsules including Umezu polyphenols (UP), polyphenols in Japanese plums, may help them to control their blood pressure (BP). The aim of this study is to evaluate the effectiveness of UP on BP and its safety. METHODS: A total of 15 healthy workers without antihypertensive medication who had some concerns about their BP, preferably normal-high BP or hypertension level 1, were randomized in a double-blind manner into UP ingesting and placebo groups. Each subject was instructed to take four capsules daily for 12 weeks (daily UP dose, 800 mg for the UP ingesting group; and 0 mg for the placebo group). These subjects were followed for 12 weeks, and their BP both at home and at the examination site, as well as self-perceived quality-of-life outcomes and possible side effects, was monitored during that period. Group × time interactions on BP changes were examined. RESULTS: All of the 15 subjects completed the 12-week intervention trial. The BP changes did not significantly differ between the UP ingesting and placebo groups, neither at the examination site nor at home. But during the study period, no adverse effects were observed. CONCLUSIONS: No remarkable effect of UP on BP was observed. However, a higher dose of UP was confirmed safe and high in adherence in this 12-week randomized controlled trial. Its effect on BP and other outcomes shall be confirmed in a larger sample.

Phenolics profile of mume, Japanese apricot (Prunus mume Sieb. et Zucc.) fruit.

The fruit of mume, Japanese apricot (Prunus mume Sieb. et Zucc.), was evaluated for its phenolics content, high performance liquid chromatography (HPLC) profile and antioxidative activities. The phenolics content of mume fruit was relatively high, the flesh of fully matured fruit containing up to 1% of phenolics on a dry weight basis. Reflecting such a high content of phenolics, the ORAC (oxygen radical absorbance capacity) value for mume fruit flesh showed high values, ranging from 150 to 320 µmol/g Trolox equivalent, depending upon the stage of maturation. 5-O-Caffeoylqunic acid (chlorogenic acid), 3-O-caffeoylquinic acid and tetra-O-acylated sucrose-related compounds were isolated from the flesh of mume fruit, although many unknown peaks were also apparent in the HPLC chromatogram. An alkali hydrolysate comprised four main phenolic acids, caffeic acid, cis/trans-p-coumaric acid and ferulic acid. No flavonoids were observed in the analysis. These results suggest that the majority of phenolics in mume fruit were hydroxycinnamic acid derivatives.

The Oral Administration of Honey Recovers the Function of the Small Intestine for Digestion and Absorption of Nutrients in a Rat Model of Total Parenteral Nutrition.

The oral administration of pure monosaccharides is effective for improving intestinal function such as nutrient digestion and absorption. However, day-to-day diets tend not to include high purity monosaccharides for intestinal health. Honey possesses large amounts of monosaccharides including glucose and fructose in the same ratio. In this study, we have evaluated the nutritional properties of honey and examined the effects of its oral ingestion on the recovery of intestinal function in the total parenteral nutrition (TPN) rat model. It was observed that honey remarkably recovered the function of the small intestine including the villous morphology, nutrient digestion, and absorption capabilities. In particular, the expression of disaccharidase was significantly enhanced by the ingestion of honey after TPN treatment. Therefore, oral intake of honey is effective in recovering and maintaining small intestinal functions and can potentially be used as a supplement for promoting small intestinal function recovery.

d-Allose is absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) in the rat small intestine.

d-Allose is the C3 epimer of d-glucose and has been reported to have beneficial health effects. The transporter mediating intestinal transport of d-allose is unknown. We examined whether d-allose is absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) as well as via glucose transporter type 5 (GLUT5) using rats. For examination of absorption via SGLT1, KGA-2727, an SGLT1-specific inhibitor, and d-allose were orally administered. KGA-2727 blocked the increase of plasma d-allose levels and suppressed them throughout the experiment (0-180 min), whereas without KGA-2727, the plasma d-allose levels peaked at around 60-90 min. For examination of absorption via GLUT5, rats were fed a high-fructose diet for 3weeks to increase the abundance and activity of GLUT5 in the small intestine. High-fructose diet-fed rats did not exhibit significant changes in the plasma d-allose levels compared to control rats fed a high-glucose diet. These results indicate that SGLT1 but not GLUT5 mediates the intestinal absorption of d-allose.

Expression of SARS-CoV-2 entry factors, electrolyte, and mineral transporters in different mouse intestinal epithelial cell types.

Angiotensin-converting enzyme 2 (ACE2) and transmembrane proteases (TMPRSS) are multifunctional proteins required for SARS-CoV-2 infection or for amino acid (AA) transport, and are abundantly expressed in mammalian small intestine, but the identity of the intestinal cell type(s) and sites of expression are unclear. Here we determined expression of SARS-CoV-2 entry factors in different cell types and then compared it to that of representative AA, electrolyte, and mineral transporters. We tested the hypothesis that SARS-CoV-2, AA, electrolyte, and mineral transporters are expressed heterogeneously in different intestinal cell types by making mouse enteroids enriched in enterocytes (ENT), goblet (GOB), Paneth (PAN), or stem (ISC) cells. Interestingly, the expression of ACE2 was apical and modestly greater in ENT, the same pattern observed for its associated AA transporters B0 AT1 and SIT1. TMPRSS2 and TMPRSS4 were more highly expressed in crypt-residing ISC. Expression of electrolyte transporters was dramatically heterogeneous. DRA, NBCe1, and NHE3 were greatest in ENT, while those of CFTR and NKCC1 that play important roles in secretory diarrhea, were mainly expressed in ISC and PAN that also displayed immunohistochemically abundant basolateral NKCC1. Intestinal iron transporters were generally expressed higher in ENT and GOB, while calcium transporters were expressed mainly in PAN. Heterogeneous expression of its entry factors suggests that the ability of SARS-CoV-2 to infect the intestine may vary with cell type. Parallel cell-type expression patterns of ACE2 with B0 AT1 and SIT1 provides further evidence of ACE2's multifunctional properties and importance in AA absorption.

Potential hypotensive effects of Umezu polyphenols: a 14-week community-based, double-masked and placebo-controlled trial.

OBJECTIVE: To evaluate blood pressure (BP)-lowering effects of Umezu polyphenols, polyphenols contained in Japanese plums, in a community-based sample by double-masked and placebo-controlled design. METHODS: Seventy-two Japanese community-dwellers who were interested in prevention or control of their BP (preferably high-normal BP or grade I hypertension) but without antihypertensive medication were randomized into Umezu polyphenols or placebo groups. Each subject took 800 mg/day of Umezu polyphenols or placebo for 12 weeks, followed by a 2-week washout period. Their home and office BP were monitored for 14 weeks in a double-masked manner. We analyzed 56 subjects who met the inclusion criteria. RESULTS: Home BP increased gradually in both the groups during the intervention period, while diastolic office BP insignificantly decreased in the intervention group. During the washout period, home systolic BP in the morning elevated only in the intervention group. CONCLUSIONS: The study failed to collect consistent evidence of a clear persistent hypotensive effect of Umezu polyphenols.

[Effects of Medium Chain Triglycerides Intake on Lipid Metabolism and Intestinal Disaccharidase Activities in Rats].

It has been reported that medium-chain triglyceride (MCT) have various physiological functions, such as anti-obesity and hypolipidemic effects. They can also elicit increased disaccharidase activity and intestinal cell proliferation. However, a meta-analysis of randomized controlled trials, comparing the effects of MCT on weight loss and body composition, detected commercial bias. Additional research on the physiological functions is needed in order to have conclusive evidence. Thus, we sought to evaluate the various functions of MCT by conducting a feeding study in rats. Rats fed a diet containing 15% (w/w) MCT, had significantly lower visceral fat weight, plasma and liver lipid concentrations; they had significantly higher intestinal maltase and glucoamylase activities; and they had a greater number of Ki-67 positive cells/crypt, compared to the rats fed a diet containing 15% (w/w) lard. The effects of a diet containing 5% (w/w) MCT was observed only for plasma cholesterol levels and the number of Ki-67 positive cells/crypt; in which some results were found to be inconsistent with previous reports. These results indicate that physiological functions of MCT are numerous and need to be confirmed by additional research.

Urinary excretion rate and bioavailability of chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid in non-fasted rats maintained under physiological conditions.

Hydroxycinnamic acids (HAs) are one of the major classes of phenolic compounds and epidemiological studies have suggested that they have beneficial health effects. This study aimed to determine the urinary excretion rate of chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid in non-fasted rats and to estimate their bioavailability under physiological conditions. Previous studies have primarily used fasted animals, which exhibit severe changes in various physiological processes. Furthermore, the food matrix can affect HA bioavailability. Thus, our studies using non-fasted rats under physiological conditions may allow for a more accurate determination of both the HA urinary excretion rate and the bioavailability of HAs. HAs were successively gavaged to rats at a dose of 40 mg/kg body weight (BW) with a wash-out period of one week. The rats were fed the AIN-93M diet throughout the experiment. The urine was collected at time intervals of 0-6 h, 6-24 h, and 24-48 h after HA administration. Ingested HAs, except chlorogenic acid, were primarily excreted in the urine within 0-6 h as free forms or conjugated (glucuronidated and/or sulfated) forms. The majority of the ingested chlorogenic acid was detected in the urine at 6-24 h or 24-48 h as caffeic acid, p-coumaric acid, ferulic acid, and their conjugates. The total urinary excretion rate (% of the dose) at 48 h was ferulic acid (73.2%) > caffeic acid (61.6%) > p-coumaric acid (54.1%) >> chlorogenic acid (4.9%). The percentages of the conjugates in the urine differed amongst the rats gavaged with the individual HAs (74% for chlorogenic acid, 83% for caffeic acid, 68% for p-coumaric acid, and 96% for ferulic acid), which may be explained by their distinct bioactivities. These data reveal that caffeic acid, p-coumaric acid, and ferulic acid are much more bioavailable than chlorogenic acid, even though they are excreted more rapidly than chlorogenic acid. Our findings may provide additional insight into the health benefits of HAs and how they function in the body.

d-Allulose is a substrate of glucose transporter type 5 (GLUT5) in the small intestine.

d-Allulose has been reported to have beneficial health effects. However, the transport system(s) mediating intestinal d-allulose transport has not yet been clearly identified. The aim of this study was to investigate whether intestinal d-allulose transport is mediated by glucose transporter type 5 (GLUT5). When d-allulose alone was gavaged, plasma d-allulose levels were dramatically higher in rats previously fed fructose. This suggests enhanced intestinal d-allulose absorption paralleled increases in GLUT5 expression observed only in fructose-fed rats. When d-allulose was gavaged with d-fructose, previously observed increases in plasma d-allulose levels were dampened and delayed, indicating d-fructose inhibited transepithelial d-allulose transport into plasma. Tracer D-[14C]-fructose uptake rate was reduced to 54.8% in 50 mM d-allulose and to 16.4% in 50 mM d-fructose, suggesting d-allulose competed with D-[14C]-fructose and the affinity of d-allulose for GLUT5 was lower than that of d-fructose. GLUT5 clearly mediates, likely at lower affinity relative to d-fructose, intestinal d-allulose transport.

Application of shielded column liquid chromatography for determination of sulfamonomethoxine, sulfadimethoxine, and their N4-acetyl metabolites in milk.

A hazardous-chemical free method for simultaneous determination of sulfamonomethoxine (SMM), sulfadimethoxine (SDM), and their N4-acetyl metabolites in raw milk using shielded column liquid chromatography is developed. The target analytes are extracted by mixing with ethanol-acetic acid (97:3, v/v) followed by centrifugation. The procedure uses a Hisep shielded hydrophobic phase (SHP) column, isocratic elution with 0.1% acetic acid solution (pH 3.1, in water)-ethanol (75:25, v/v), and a photo-diode array detector. Average recoveries from samples spiked at 25-500 ng/ml for each drug were >81% with relative standard deviations within 5%. The limits of quantitation were <25 ng/ml.

An association analysis between mitochondrial DNA A10398G polymorphism and temperament in Japanese young adults.

The mitochondrial (mt) DNA C5178A and A10398G polymorphisms have been reported to be associated with mental disorders such as bipolar disorder. However, the effects of these polymorphisms on temperament in healthy people are poorly understood. Evaluating healthy subjects can have the advantage of providing new strategies for maintaining psychological health and preventing mental illness. We examined the association between mtDNA polymorphisms and temperament in Japanese students. There was no significant difference in examined temperament when analysed by genotypes, 5178-10398 haplotypes, or sex. The subgroup analysis based on sex indicated that there was an interactive effect of the mtDNA A10398G polymorphism and sex on anxiety and obsession. This finding is preliminary and cannot exclude the possibility of false-positive due to small sample size (144 subjects) and multiple statistical testing. Further studies involving a larger sample size or other ethnic groups are necessary to confirm that mtDNA A10398G polymorphism can be a genetic factor for temperament.

Simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N(4)-acetyl metabolites with gradient liquid chromatography in chicken plasma, tissues, and eggs.

A simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N(4)-acetyl metabolites in chicken plasma, muscle, liver, and eggs using gradient high-performance liquid chromatography (HPLC) with a photo-diode array detector is developed. All the compounds are extracted by a handheld ultrasonic homogenizer with ethanol followed by centrifugation. The separation is performed by a reversed-phase C4 column with a gradient elution (ethanol:1% (v/v) acetic acid, v/v; 10:90-->20:80). Average recoveries from samples spiked at 0.1-1.0mugg(-1) or mugml(-1) for each drug were >90% with relative standard deviations within 4%. The limits of quantitation were <30ngg(-1) or ngml(-1).