Development of New Targeted Inulin Complex Nanoaggregates for siRNA Delivery in Antitumor Therapy.

Here, a novel strategy of formulating efficient polymeric carriers based on the already described INU-IMI-DETA for gene material whose structural, functional, and biological properties can be modulated and improved was successfully investigated. In particular, two novel derivatives of INU-IMI-DETA graft copolymer were synthesized by chemical functionalisation with epidermal growth factor (EGF) or polyethylenglycol (PEG), named INU-IMI-DETA-EGF and INU-IMI-DETA-PEG, respectively, in order to improve the performance of already described "inulin complex nanoaggregates" (ICONs). The latter were thus prepared by appropriately mixing the two copolymers, by varying each component from 0 to 100 wt% on the total mixture, named EP-ICONs. It was seen that the ability of the INU-IMI-DETA-EGF/INU-IMI-DETA-PEG polymeric mixture to complex siGL3 increases with the increase in the EGF-based component in the EP-ICONs and, for each sample, with the increase in the copolymer:siRNA weight ratio (R). On the other hand, the susceptibility of loaded siRNA towards RNase decreases with the increase in the pegylated component in the polymeric mixture. At all R values, the average size and the zeta potential values are suitable for escaping from the RES system and suitable for prolonged intravenous circulation. By means of biological characterisation, it was shown that MCF-7 cells are able to internalize mainly the siRNA-loaded into EGF-decorated complexes, with a significant difference from ICONs, confirming its targeting function. The targeting effect of EGF on EP-ICONs was further demonstrated by a competitive cell uptake study, i.e., after cell pre-treatment with EGF. Finally, it was shown that the complexes containing both EGF and PEG are capable of promoting the internalisation and therefore the transfection of siSUR, a siRNA acting against surviving mRNA, and to increase the sensitivity to an anticancer agent, such as doxorubicin.

Deletion of the Imprinted Phlda2 Gene Increases Placental Passive Permeability in the Mouse.

Genomic imprinting, an epigenetic phenomenon that causes the expression of a small set of genes in a parent-of-origin-specific manner, is thought to have co-evolved with placentation. Many imprinted genes are expressed in the placenta, where they play diverse roles related to development and nutrient supply function. However, only a small number of imprinted genes have been functionally tested for a role in nutrient transfer capacity in relation to the structural characteristics of the exchange labyrinthine zone. Here, we examine the transfer capacity in a mouse model deficient for the maternally expressed Phlda2 gene, which results in placental overgrowth and a transient reduction in fetal growth. Using stereology, we show that the morphology of the labyrinthine zone in Phlda2-/+ mutants is normal at E16 and E19. In vivo placental transfer of radiolabeled solutes 14C-methyl-D-glucose and 14C-MeAIB remains unaffected at both gestational time points. However, placental passive permeability, as measured using two inert hydrophilic solutes (14C-mannitol; 14C-inulin), is significantly higher in mutants. Importantly, this increase in passive permeability is associated with fetal catch-up growth. Our findings uncover a key role played by the imprinted Phlda2 gene in modifying placental passive permeability that may be important for determining fetal growth.

Does "Birth" as an Event Impact Maturation Trajectory of Renal Clearance via Glomerular Filtration? Reexamining Data in Preterm and Full-Term Neonates by Avoiding the Creatinine Bias.

Glomerular filtration rate (GFR) is an important measure of renal function. Various models for its maturation have recently been compared; however, these have used markers, which are subject to different renal elimination processes. Inulin clearance data (a purer probe of GFR) collected from the literature were used to determine age-related changes in GFR aspects of renal drug excretion in pediatrics. An ontogeny model was derived using a best-fit model with various combinations of covariates such as postnatal age, gestational age at birth, and body weight. The model was applied to the prediction of systemic clearance of amikacin, gentamicin, vancomycin, and gadobutrol. During neonatal life, GFR increased as a function of both gestational age at birth and postnatal age, hence implying an impact of birth and a discrepancy in GFR for neonates with the same postmenstrual age depending on gestational age at birth (ie, neonates who were outside the womb longer had higher GFR, on average). The difference in GFR between pre-term and full-term neonates with the same postmenstrual age was negligible from beyond 1.25 years. Considering both postnatal age and gestational age at birth in GFR ontogeny models is important because postmenstrual age alone ignores the impact of birth. Most GFR models use covariates of body size in addition to age. Therefore, prediction from these models will also depend on the change in anthropometric characteristics with age. The latter may not be similar in various ethnic groups, and this makes the head-to-head comparison of models very challenging.

Fermentation Kinetics of Selected Dietary Fibers by Human Small Intestinal Microbiota Depend on the Type of Fiber and Subject.

SCOPE: An underexplored topic is the investigation of health effects of dietary fibers via modulation of human small intestine (SI) microbiota. A few previous studies hint at fermentation of some dietary fibers in the distal SI of humans and pigs. Here the potential of human SI microbiota to degrade dietary fibers and produce metabolites in vitro is investigated. METHODS AND RESULTS: Fructans, galacto-oligosaccharides, lemon pectins, and isomalto/malto-polysaccharides are subjected to in vitro batch fermentations inoculated with ileostomy effluent from five subjects. Fiber degradation products, formation of bacterial metabolites, and microbiota composition are determined over time. Galacto- and fructo-oligosaccharides are rapidly utilized by the SI microbiota of all subjects. At 5h of fermentation, 31%-82% of galacto-oligosaccharides and 29%-89% fructo-oligosaccharides (degree of polymerization DP4-8) are utilized. Breakdown of fructo-oligosaccharides/inulin DP ≥ 10, lemon pectin, and iso-malto/maltopolysaccharides only started after 7h incubation. Degradation of different fibers result in production of mainly acetate, and changed microbiota composition over time. CONCLUSION: Human SI microbiota have hydrolytic potential for prebiotic galacto- and fructo-oligosaccharides. In contrast, the higher molecular weight fibers inulin, lemon pectin, and iso-malto/maltopolysaccharides show slow fermentation rate. Fiber degradation kinetics and microbiota responses are subject dependent, therefore personalized nutritional fiber based strategies are required.

Preparation and release properties of arbutin imprinted inulin/polyvinyl alcohol biomaterials.

The main objective of this work was to prepare inulin (INL)/polyvinyl alcohol (PVA) biomaterials imprinted with arbutin (AR) as the target drug. INL from Jerusalem artichoke flour was extracted with hot water extraction method. INL/PVA biomaterials were synthesized with a casting method and a UV curing. The optimal UV curing time and sodium benzoate content were about 10 min and 0.1 wt%, respectively. The biomaterials were characterized by SEM and FT-IR analysis. Mechanical properties of prepared AR imprinted biomaterials were also investigated. AR release was examined with changes of pH at 36.5 °C. The AR release ratio was also investigated using artificial skin. It was found that AR was released constantly for 40 min. Results of drug release mechanism indicated that AR release followed the Fickian diffusion behavior, whereas drug release using artificial skin followed the non-Fickian diffusion behavior. Tyrosinase inhibitory (%) for AR imprinted biomaterials with/without the addition of GL were 58.8% and 79.2%, respectively.

Fermentation of Chicory Fructo-Oligosaccharides and Native Inulin by Infant Fecal Microbiota Attenuates Pro-Inflammatory Responses in Immature Dendritic Cells in an Infant-Age-Dependent and Fructan-Specific Way.

SCOPE: Inulin-type fructans are commonly applied in infant formula to support development of gut microbiota and immunity. These inulin-type fructans are considered to be fermented by gut microbiota, but it is unknown how fermentation impacts immune modulating capacity and whether the process of fermentation is dependent on the infant's age. METHODS AND RESULTS: The in vitro fermentation of chicory fructo-oligosaccharides (FOS) and native inulin are investigated using pooled fecal inocula of two- and eight-week-old infants. Both inocula primarily utilize the trisaccharides in FOS, while they almost completely utilize native inulin with degree of polymerization (DP) 3-8. Fecal microbiota of eight-week-old infants degrades longer chains of native inulin up to DP 16. This correlates with a higher abundance of Bifidobacterium and higher production of acetate and lactate after 26 h of fermentation. Fermented FOS and native inulin attenuate pro-inflammatory cytokines produced by immature dendritic cells (DCs), but profiles and magnitude of attenuation are stronger with native inulin than with FOS. CONCLUSION: The findings demonstrate that fermentation of FOS and native inulin is dependent on the infant's age and fructan structure. Fermentation enhances attenuating effects of pro-inflammatory responses in DCs, which depend mainly on microbial metabolites formed during fermentation.

Inulin as carriers for renal targeting delivery of ferulic acid.

Inulin (IN), as a classic diagnostic for determination of glomerular filtration rate, reached high concentration in kidney. Introducing drug into IN derivatives may be a new method to target kidney for drug delivery. To test the hypothesis, ferulic acid (FeA) was conjugated into IN by ester bond and amide bond (ethylenediamine as spacer), respectively, and the two FeA-IN conjugations, inulin ferulate (IN-FeA) and inulin ethylenediamine ferulate (IN-EDA-FeA) were obtained. NMR spectrum was involved to characterize the conjugations. The FeA in vitro release profiles were tested in mice plasma and renal homogenate. Finally, the biodistribution test was performed to evaluate their renal-targeting ability. Both IN-FeA and IN-EDA-FeA showed a higher release rate of FeA in renal homogenate than in mouse plasma suggesting the conjugates are relatively stable in plasma and more likely FeA release in kidney. The renal area under the curve (AUC) for IN-FeA and IN-EDA-FeA were 539.6 ± 107.9 and 558.5 ± 131.6 µg h/mL, respectively, which were 4.47 and 4.62 times of 120.8 ± 18.1 µg h/mL for free FeA. Meanwhile, significant smaller FeA accumulation in other organs was observed. These data indicated that IN-FeA and IN-EDA-FeA effectively targeted kidney for FeA delivery.

Role of maltodextrin and inulin as encapsulating agents on the protection of oleuropein during in vitro gastrointestinal digestion.

Olive leaves extract (OLE) was spray-dried with maltodextrin (MD) or inulin (IN) to study the evolution of oleuropein (OE) during in vitro gastrointestinal digestion, its bioaccessibility and potential bioavailability. In the case of OLE-MD, OE was partially degraded in gastric and intestinal conditions; whereas in OLE-IN, OE was released under gastric conditions and partially degraded under intestinal conditions. In both cases, the encapsulation of OLE led to higher OE contents at the end of digestion, compared with non-encapsulated OLE, suggesting a protective role of the polysaccharides by the formation of non-covalent polysaccharides-OE complexes. OE bioaccessibility was ten times higher (p ≤ 0.05) in OLE-MD and OLE-IN than in non-encapsulated OLE. However, OE potential bioavailability, evaluated by tangential filtration, was not detected. Encapsulation technology and the encapsulant agent used may determine the release of the encapsulated compounds at a specific-site and their effect on health.

Impact of dietary ingredients on the interpretation of various fecal parameters in rats fed inulin.

In the present study, the influences of diets (i.e. chow and AIN-93 diets) on the interpretation of various fecal parameters including viable microbiota, moisture, weight, and short-chain fatty acids in rats fed different amounts of inulin (0.5-2 g/kg). Eight groups of rats (n = 8/group) were fed, for 4 weeks, chow or AIN-93 diets with or without inulin supplementation. Fecal samples were analyzed for different fecal parameters. After a 2-week adaptation, apparent differences in some fecal parameters were observed between the chow and AIN-93 diet groups. Throughout the 4-week intervention period, significantly (p < 0.05) higher Lactobacillus spp. counts, fecal moisture (∼2.7-fold), and fecal weight (∼5.8-fold) were observed with chow diet over AIN-93 diet. More specifically, significant elevations in the levels of Bifidobacterium spp., Lactobacillus spp., fecal moisture, and fecal weight could be observed at low-dose (0.5 g/kg) of inulin in chow diet groups, while most of these changes could merely be seen at medium-dose (1 g/kg) in AIN-93 diet groups. These results demonstrated that the choice of experimental diets would affect the comparison of fecal parameters as well as the interpretation of effective dosage of prebiotic in intestinal health assessments.

Oral Delivery of Probiotics Using pH-Sensitive Phthalyl Inulin Tablets.

Probiotics show low cell viability after oral administration because they have difficulty surviving in the stomach due to low pH and enzymes. For the oral delivery of probiotics, developing a formula that protects the probiotic bacteria from gastric acidity while providing living cells is mandatory. In this study, we developed tablets using a new pH-sensitive phthalyl inulin (PI) to protect probiotics from gastric conditions and investigated the effects of different compression forces on cell survival. We made three different tablets under different compression forces and measured survivability, disintegration time, and kinetics in simulated gastric-intestinal fluid. During tableting, there were no significant differences in probiotic viability among the different compression forces although disintegration time was affected by the compression force. A higher compression force resulted in higher viability in simulated gastric fluid. The swelling degree of the PI tablets in simulated intestinal fluid was higher than that of the tablets in simulated gastric fluid due to the pH sensitivity of the PI. The probiotic viability formulated in the tablets was also higher in acidic gastric conditions than that for probiotics in solution. Rapid release of the probiotics from the tablet occurred in the simulated intestinal fluid due to the pH sensitivity. After 6 months of refrigeration, the viability of the PI probiotics was kept. Overall, this is the first study to show the pH-sensitive properties of PI and one that may be useful for oral delivery of the probiotics.

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs. To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules. Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error. We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity. In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Development and application of bio-sample quantification to evaluate stability and pharmacokinetics of inulin-type fructo-oligosaccharides from Morinda Officinalis.

Inulin-type fructooligosaccharides (FOS) purified from Morinda Officinalis, with degrees of polymerization (DP) from 3 to 9, have been approved in China as an oral prescribed drug for mild and moderate depression episode, while the stability and oral absorption of this FOS mixtures are largely unknown. As the main active component and quality control marker for above FOS, DP5 was selected as the representative FOS in this study. Desalting method by ion exchange resin was developed to treat bio-sample, followed by separation and quantification by high performance liquid chromatography-charged aerosol detector. Results showed that the DP5 was stepwisely hydrolyzed in simulated gastric fluid and gut microbiota, while maintained stable in intestinal fluid. DP5 has poor permeability across Caco-2 monolayer with Papp of 5.22 × 10-7 cm/s, and very poor oral absorption with bioavailability of (0.50 ±â€¯0.12)% in rat. In conclusion, FOS in Morinda Officinalis demonstrated poor chemical stability in simulated gastric fluid and human gut microbiota, and low oral absorption in rats.

Development and validation of a physiology-based model for the prediction of pharmacokinetics/toxicokinetics in rabbits.

The environmental fates of pharmaceuticals and the effects of crop protection products on non-target species are subjects that are undergoing intense review. Since measuring the concentrations and effects of xenobiotics on all affected species under all conceivable scenarios is not feasible, standard laboratory animals such as rabbits are tested, and the observed adverse effects are translated to focal species for environmental risk assessments. In that respect, mathematical modelling is becoming increasingly important for evaluating the consequences of pesticides in untested scenarios. In particular, physiologically based pharmacokinetic/toxicokinetic (PBPK/TK) modelling is a well-established methodology used to predict tissue concentrations based on the absorption, distribution, metabolism and excretion of drugs and toxicants. In the present work, a rabbit PBPK/TK model is developed and evaluated with data available from the literature. The model predictions include scenarios of both intravenous (i.v.) and oral (p.o.) administration of small and large compounds. The presented rabbit PBPK/TK model predicts the pharmacokinetics (Cmax, AUC) of the tested compounds with an average 1.7-fold error. This result indicates a good predictive capacity of the model, which enables its use for risk assessment modelling and simulations.

Maternal Hypercholesterolemia Associated with Nicotine Exposure in Adulthood May Induce Kidney Injury in Male Rats if Hypomagnesemia Occurs.

BACKGROUND/AIMS: Maternal hypercholesterolemia is a risk factor to renal injury in rat pups at adulthood, especially if they feed a cholesterol-enriched diet after weaning. However, the renal function of male pups of dams with hypercholesterolemia (PH) that were fed a regular chow from weaning to adulthood needs investigation, particularly those exposed to an adverse risk such as nicotine. METHODS: We evaluated the renal function of PH animals and we compared the data with those found in male pups of control dams (PC) at 3- and 6-month-old by inulin clearance. Moreover, we investigated the effect of nicotine treatment for 8 days in both PH and PC animals at 6 months old via metabolic function studies and by renal histological analysis. RESULTS: Inulin clearance and other renal function parameters were similar in PH and PC animals at 3 and 6 months old. Nevertheless, the PH group showed significant differences with regard to histological analysis despite a similar number of glomeruli. The glomerular area of PH animals was significantly smaller than that measured in PC animals, and the fractional interstitial area was significantly larger in PH animals than that measured in PC animals at 3 months old. With regard to nicotine treatment, we observed a trend for a reduction in creatinine clearance in both PC and PH groups, but only PH animals showed hypomagnesemia and the highest fractional interstitial area. CONCLUSIONS: The offspring exposed to a high cholesterol milieu during intrauterine and neonatal life may show a silent kidney injury at adulthood that may be aggravated by nicotine exposure if hypomagnesemia occurs.

Validation of a routine two-sample iohexol plasma clearance assessment of GFR and an evaluation of common endogenous markers in a rat model of CKD.

Endogenous markers of kidney function are insensitive to early declines in glomerular filtration rate (GFR) and in rodent models, validated, practical alternatives are unavailable. In this study, we determined GFR by modeling the plasma clearance of two compounds, iohexol and inulin, and compared the findings to common endogenous markers. All plasma clearance methods of both iohexol and inulin detected a decline in renal function weeks prior to any increase in endogenous marker. Iohexol plasma clearance and inulin plasma clearance had a very high agreement and minimal bias when using 12-sample models. However, only iohexol could be accurately simplified to a two-sample, one-compartment estimation strategy. Following an IV injection of low-dose iohexol and two timed blood samples at 30 and 90 min, one can accurately approximate a complex 12-sample strategy of plasma clearance. This method is simple enough to use in routine, longitudinal analysis of larger cohort animal studies.

Drinking and water permeability in the Pacific hagfish, Eptatretus stoutii.

Hagfish are osmoconformers, maintaining an internal osmolality that matches their seawater habitats. Hagfish would, therefore, appear to have no physiological need to drink, but previous studies are equivocal regarding whether drinking in hagfish occurs. The current study addressed this knowledge gap, by examining drinking and water permeability in the Pacific hagfish, Eptatretus stoutii. One-third of analysed hagfish were shown to accumulate radiolabelled drinking rate markers (tritiated inulin and polyethylene glycol-4000) in their gut tissues; however, this was attributed to the presence of markers in the blood perfusing the digestive tract, following absorption through paracellular pathways at the gill. No accumulation of marker was observed in hagfish subjected to more dilute (75% seawater) or more concentrated (125% seawater) media. Diffusive water efflux, measured by tritiated water washout, was shown to be very high, with 50% of body water exchanged within 14 to 16 min, depending on exposure salinity. In full-strength seawater, the total exchangeable pool of water was 78% of hagfish mass. We conclude that hagfish do not drink, and their high water permeability is likely to result in rapid osmotic equilibration under circumstances where perturbations may occur.

Design, synthesis and evaluation of biotin decorated inulin-based polymeric micelles as long-circulating nanocarriers for targeted drug delivery.

Here, long-circulating behaviors of Inulin-based nanomicelles are demonstrated for the first time in vivo. We show the synthesis and evaluation of biotin (BIO)-decorated polymeric INVITE micelles constituted of substances of natural origin, Inulin (INU) and Vitamin E (VITE), as long-circulating carriers for receptor-mediated targeted drug delivery. The resulting INVITE or INVITE-BIO micelles, nanometrically sized, did not reveal any cytotoxicity after 24h of incubation with Caco-2 cells. Moreover, in vitro studies on Caco-2 cells monolayers indicated that the transport of INVITE-BIO micelles was faster than surface unmodified INVITE micelles. In vivo optical imaging studies evidenced that, upon intravenous administration, INVITE-BIO micelles were quantitatively present in the body up to 48h. Instead, after oral administration, the micelles were not found in the systemic circulation but eliminated with the normal intestinal content. In conclusion, INVITE-BIO micelles may enhance drug accumulation in tumor-cells over-expressing the receptor for biotin through receptor mediated endocytosis.

Influencia del suplemento con inulina enriquecida con fructooligosacáridos sobre el contenido y la densidad mineral ósea tras el parto y la lactación en ratas / Influence of supplementation with oligofructose-enriched inulin on bone mineral content and density after delivery and lactation in rats

Introducción: la gestación y lactancia están relacionadas con pérdidas temporales en la densidad mineral ósea (DMO) materna. Una suplementación con calcio podría resultar beneficiosa para evitar la pérdida de masa ósea del esqueleto materno. Otros nutrientes como los prebióticos han sido identificados como responsables de un incremento en la absorción de minerales, pudiendo condicionar la mineralización ósea. Objetivo: estudiar el efecto de la suplementación de la dieta materna con el prebiótico inulina enriquecida con oligofructosa, durante la gestación y la lactancia sobre el contenido mineral óseo (CMO) y la DMO al final del periodo de lactancia. Métodos: las ratas gestantes fueron alimentadas con dieta estándar (grupo CC), dieta fortificada en calcio (grupo Ca) o enriquecida con el prebiótico inulina enriquecida con oligofructosa (grupo Pre) hasta el final del periodo de lactancia. Posteriormente se evaluó el CMO y DMO por absorciometría de rayos X (DEXA) y el pH del contenido cecal. Resultados: en términos generales, el grupo Pre presenta los mayores valores absolutos de CMO y DMO de entre los tres grupos, siendo en la tibia significativamente diferentes en los grupos CC y Pre frente al grupo Ca. El pH del contenido cecal del grupo Pre es signifi cativamente inferior al de los grupos CC y Ca. Conclusión: la suplementación con inulina enriquecida con oligofructosa, en condiciones nutricionales no deficientes en calcio, durante la gestación y la lactancia, ejerce una protección del esqueleto materno en las ratas y puede ser considerada como una estrategia nutricional para proteger la masa ósea materna en el periodo perinatal (AU)

Introduction: Pregnancy and lactation are related with temporary decreases in maternal bone mineral density (BMD). Calcium supplementation could be beneficial to prevent bone loss of maternal skeleton. Other nutrients, such as prebiotics have showed to produce an increase of the mineral absorption and therefore affecting bone mineralization. Objective: To study the effect of maternal diet supplementation with prebiotic oligofructose-enriched inulin during gestation and lactation on the maternal bone mineral content (BMC) and BMD at the end of lactation. Methods: Pregnant rats were fed with standard diet (CC group), calcium fortified diet (Ca group) or with prebiotic oligofructose-enriched inulin supplemented diet until the end of the lactation period. At weaning, bone mineral content (BMC) and BMD were determined by dual-energy X-ray absorptiometry and the pH of the cecal content was also determined. Results: In absolute terms, the highest BMD and BMC were found in the Pre group as compared with the other two groups being significant in the tibia when compared Pre group and CC group with Ca group. The pH of the cecal content in the Pre group was also significantly lower as compared with the other two groups. Conclusion: Prebiotic oligofructose-enriched inulin supplementation, in calcium no-deficient conditions, during gestation and lactation exerts a protection on maternal skeleton during pregnancy and lactation in the rats and could be considered as a plausible nutritional option for protecting maternal bone mass during these periods (AU)

Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions, gut barrier dysfunction and liver damage in mice.

BACKGROUND: Disruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury. METHODS: Wild type (WT) and occludin deficient (Ocln(-/-)) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers. RESULTS: Ethanol feeding significantly reduced body weight gain in Ocln(-/-) mice. Ethanol increased inulin permeability in colon of both WT and Ocln(-/-) mice, but the effect was 4-fold higher in Ocln(-/-) mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and ß-catenin from the junctions and elevated TLR4, which was more severe in Ocln(-/-) mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln(-/-) mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln(-/-) mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln(-/-) mice. CONCLUSION: This study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.

Bioavailability of insulin detemir and human insulin at the level of peripheral interstitial fluid in humans, assessed by open-flow microperfusion.

AIMS: To find an explanation for the lower potency of insulin detemir observed in humans compared with unmodified human insulin by investigating insulin detemir and human insulin concentrations directly at the level of peripheral insulin-sensitive tissues in humans in vivo. METHODS: Euglycaemic-hyperinsulinaemic clamp experiments were performed in healthy volunteers. Human insulin was administered i.v. at 6 pmol/kg/min and insulin detemir at 60 pmol/kg/min, achieving a comparable steady-state pharmacodynamic action. In addition, insulin detemir was doubled to 120 pmol/kg/min. Minimally invasive open-flow microperfusion (OFM) sampling methodology was combined with inulin calibration to quantify human insulin and insulin detemir in the interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissue. RESULTS: The human insulin concentration in the ISF was ∼115 pmol/l or ∼30% of the serum concentration, whereas the insulin detemir concentration in the ISF was ∼680 pmol/l or ∼2% of the serum concentration. The molar insulin detemir interstitial concentration was five to six times higher than the human insulin interstitial concentration and metabolic clearance of insulin detemir from serum was substantially reduced compared with human insulin. CONCLUSIONS: OFM proved useful for target tissue measurements of human insulin and the analogue insulin detemir. Our tissue data confirm a highly effective retention of insulin detemir in the vascular compartment. The higher insulin detemir relative to human insulin tissue concentrations at comparable pharmacodynamics, however, indicate that the lower potency of insulin detemir in humans is attributable to a reduced effect in peripheral insulin-sensitive tissues and is consistent with the reduced in vitro receptor affinity.