Inhibition of Intestinal Thiamin Transport in Rat Model of Sepsis.
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ABSTRACT: Thiamin deficiency is highly prevalent in patients with sepsis, but the mechanism by which sepsis induces thiamin deficiency is unknown. This study aimed to determine the influence of various severity of sepsis on carrier-mediated intestinal thiamin uptake, level of expressions of thiamin transporters (thiamin transporter-1 and thiamin transporter-2), and mitochondrial thiamin pyrophosphate transporter.Randomized controlled study.Research laboratory at a Veterans Affairs Medical Center.Twenty-four Sprague-Dawley rats were randomized into controls, mild, moderate, and severe sepsis with equal number of animals in each group.Sepsis was induced by cecal ligation and puncture with the cecum ligated below the cecal valve at 25%, 50%, and 75% of cecal length, defined as severe, moderate, and mild sepsis, respectively. Control animals underwent laparotomy only.After 2 days of induced sepsis, carrier-mediated intestinal thiamin uptake was measured using [H]thiamin. Expressions of thiamin transporter-1, thiamin transporter-2, and mitochondrial thiamin pyrophosphate transporter proteins and messenger RNA were measured. Proinflammatory cytokines (interleukin-1? and interleukin-6) and adenosine triphosphate were also measured. Sepsis inhibited [H]thiamin uptake, and the inhibition was a function of sepsis severity. Both cell membrane thiamin transporters and mitochondrial thiamin pyrophosphate transporter expression levels were suppressed; also levels of adenosine triphosphate in the intestine of animals with moderate and severe sepsis were significantly lower than that of sham-operated controls.For the first time, we demonstrated that sepsis inhibited carrier-mediated intestinal thiamin uptake as a function of sepsis severity, suppressed thiamin transporters and mitochondrial thiamin pyrophosphate transporter, leading to adenosine triphosphate depletion.
<h4>Objectives</h4>Thiamin deficiency is highly prevalent in patients with sepsis, but the mechanism by which sepsis induces thiamin deficiency is unknown. This study aimed to determine the influence of various severity of sepsis on carrier-mediated intestinal thiamin uptake, level of expressions of thiamin transporters (thiamin transporter-1 and thiamin transporter-2), and mitochondrial thiamin pyrophosphate transporter.<h4>Design</h4>Randomized controlled study.<h4>Setting</h4>Research laborat ...[more]
Project description:End-stage renal disease is a worldwide health burden, but the pathogenesis of uremia-associated cognitive impairment (CI) is poorly recognized. We hypothesized that uremia brings about deficiency of thiamin and folic acid and causes CI by inducing oxidative stress. Therefore, 24 Sprague-Dawley rats were randomly divided into two groups: a 5/6 nephrectomy group (n = 12) and a sham-operated group (n = 12). The Morris water maze was used to assess the cognitive function eight weeks post-surgery, and serum levels of thiamin, folic acid and homocysteine were detected subsequently. Brain and kidney tissues were collected for pathological examination and 8-Hydroxy-2'-deoxyguanosine (8-OHdG) immunochemistry staining. Results showed that the escape latency on training days 1-2 was longer, and the time in quadrant IV on experimental day 6 was significantly shorter in 5/6 nephrectomy group. Meanwhile, the uremic rats showed decreased thiamin, folic acid and increased homocysteine. We also found the time in quadrant IV was positively correlated with thiamin and folic acid level, while negatively correlated with the blood urea nitrogen and 8-OHdG positive cell proportion. Furthermore, in 5/6 nephrectomy group, the hippocampal neuron count was significantly reduced, and a greater proportion of 8-OHdG positive cells were detected. Pretreating LPS-stimulated rat microglial cells with thiamin or folic acid in vitro alleviated the inflammatory impairment in terms of cell viability and oxidative stress. In summary, we applied a uremic rat model and proved that uremia causes serum thiamin and folic acid deficiency, homocysteine elevation, along with neuron reduction and severe oxidative stress in hippocampus, finally leading to CI.
Project description:To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects.Rats underwent fractionated X-irradiation (5 Gy×9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation.Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups.Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.
Project description:In this work, we studied the intestinal absorption of a peptide with a molecular weight of 4353 Da (MEDI7219) and a protein having a molecular weight of 11 740 Da (PEP12210) in the rat intestinal instillation model and compared their absorption to fluorescein isothiocyanate (FITC)-labeled dextrans of similar molecular weights (4 and 10 kDa). To increase the absorption of the compounds, the permeation enhancer sodium caprate (C10) was included in the liquid formulations at concentrations of 50 and 300 mM. All studied compounds displayed an increased absorption rate and extent when delivered together with 50 mM C10 as compared to control formulations not containing C10. The time period during which the macromolecules maintained an increased permeability through the intestinal epithelium was approximately 20 min for all studied compounds at 50 mM C10. For the formulations containing 300 mM C10, it was noted that the dextrans displayed an increased absorption rate (compared to 50 mM C10), and their absorption continued for at least 60 min. The absorption rate of MEDI7219, on the other hand, was similar at both studied C10 concentrations, but the duration of absorption was extended at the higher enhancer concentration, leading to an increase in the overall extent of absorption. The absorption of PEP12210 was similar in terms of the rate and duration at both studied C10 concentrations. This is likely caused by the instability of this molecule in the intestinal lumen. The degradation decreases the luminal concentrations over time, which in turn limits absorption at time points beyond 20 min. The results from this study show that permeation enhancement effects cannot be extrapolated between different types of macromolecules. Furthermore, to maximize the absorption of a macromolecule delivered together with C10, prolonging the duration of absorption appears to be important. In addition, the macromolecule needs to be stable enough in the intestinal lumen to take advantage of the prolonged absorption time window enabled by the permeation enhancer.
Project description:Food-drug interaction studies are critical to evaluate appropriate dosing, timing, and formulation of new drug candidates. These interactions often reflect prandial-associated changes in the extent and/or rate of systemic drug exposure. Physiologic and physicochemical mechanisms underlying food effects on drug disposition are well-characterized. However, biochemical mechanisms involving drug metabolizing enzymes and transport proteins remain underexplored. Several plant-derived beverages have been shown to modulate enzymes and transporters in the intestine, leading to altered pharmacokinetic (PK) and potentially negative pharmacodynamic (PD) outcomes. Commonly consumed fruit juices, teas, and alcoholic drinks contain phytochemicals that inhibit intestinal cytochrome P450 and phase II conjugation enzymes, as well as uptake and efflux transport proteins. Whereas myriad phytochemicals have been shown to inhibit these processes in vitro, translation to the clinic has been deemed insignificant or undetermined. An overlooked prerequisite for elucidating food effects on drug PK is thorough knowledge of causative bioactive ingredients. Substantial variability in bioactive ingredient composition and activity of a given dietary substance poses a challenge in conducting robust food-drug interaction studies. This confounding factor can be addressed by identifying and characterizing specific components, which could be used as marker compounds to improve clinical trial design and quantitatively predict food effects. Interpretation and integration of data from in vitro, in vivo, and in silico studies require collaborative expertise from multiple disciplines, from botany to clinical pharmacology (i.e., plant to patient). Development of more systematic methods and guidelines is needed to address the general lack of information on examining drug-dietary substance interactions prospectively.
Project description:Roux-en-Y gastric bypass (RYGB) has become the gold-standard bariatric procedure, partly because of the rapid resolution of accompanying diabetes. There is increasing evidence this is mediated by duodenal exclusion. We hypothesize that duodenal exclusion suppresses intestinal Na(+)/glucose cotransporter SGLT1-mediated glucose transport, improving glucose handling, and aimed to test this in a rodent RYGB model. Sprague-Dawley rats underwent sham procedure or duodenal exclusion by RYGB (10 cm Roux, 16 cm biliopancreatic limbs). Animals were maintained for 3 wk on a Western diet, before harvest at 10 AM, 4 PM, and 10 PM. Sections were taken from each limb for hematoxylin and eosin staining, and morphological assessment was performed. Functional glucose uptake studies, along with Western blotting and quantitative PCR, were performed on Roux limb. Histology showed morphometric changes in Roux and common limbs, with increase in villus height and crypt depth compared with BP and sham jejunum. Despite this, glucose transport was reduced by up to 68% (P < 0.001) in the Roux limb compared with sham jejunum. Normal diurnal rhythms in glucose uptake were ablated. This occurred at a posttranscriptional level, with little change in message but appearance of different weight species of Sglt1 on Western blotting. We have shown duodenal exclusion significantly influences both intestinal structure and glucose transport function, with glucose absorptive capacity reduced after RYGB. This provides a novel mechanistic explanation for some of the antidiabetic effects of RYGB.
Project description:BackgroundTo describe the effect of mechanical ventilation on diaphragm mitochondrial oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and cytochrome c oxidase activity and content, and their relationship to diaphragm strength in an experimental model of sepsis.MethodsA cecal ligation and puncture (CLP) protocol was performed in 12 rats while 12 controls underwent sham operation. Half of the rats in each group were paralyzed and mechanically ventilated. We performed blood gas analysis and lactic acid assays 6 h after surgery. Afterwards, we measured diaphragm strength and mitochondrial oxygen consumption, ATP and ROS generation, and cytochrome c oxidase activity. We also measured malondialdehyde (MDA) content as an index of lipid peroxidation, and mRNA expression of the proinflammatory interleukin-1β (IL-1β) in diaphragms.ResultsCLP rats showed severe hypotension, metabolic acidosis, and upregulation of diaphragm IL-1β mRNA expression. Compared to sham controls, spontaneously breathing CLP rats showed lower diaphragm force and increased susceptibility to fatigue, along with depressed mitochondrial oxygen consumption and ATP production and cytochrome c oxidase activity. These rats also showed increased mitochondrial ROS generation and MDA content. Mechanical ventilation markedly restored mitochondrial oxygen consumption and ATP production in CLP rats; lowered mitochondrial ROS production by the complex 3; and preserved cytochrome c oxidase activity.ConclusionIn an experimental model of sepsis, early initiation of mechanical ventilation restores diaphragm mitochondrial function.
Project description:Intestinal barrier dysfunction is associated with the occurrence and development of sepsis. Further, aerobic glycolysis plays an essential role in inflammation and cell death. This study is aimed at investigating the protective effect and mechanism of PFKFB3 inhibition on intestinal barrier dysfunction in sepsis mice. Sepsis mouse models were established by cecal ligation and puncture (CLP) in wild-type mice and Gsdmd-/- mice. The results showed that the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in the small intestines was significantly upregulated in sepsis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), the specific inhibitor of PFKFB3, and Gsdmd gene knockout significantly inhibited the inflammatory response and cell death caused by sepsis, thus alleviating intestinal damage and barrier dysfunction. 3PO was also shown to significantly inhibit oxidative stress and NLRP3/caspase-1/GSDMD-dependent cell pyroptosis in the small intestines. The in vitro studies revealed that 3PO reduced NLRP3/caspase-1/GSDMD-dependent cell pyroptosis by inhibiting ROS. Taken together, our results suggest that PFKFB3 is involved in inflammation, oxidative stress, and pyroptosis during sepsis and enhances intestinal damage, which may provide important clues about the potential targets to be exploited in this highly lethal disease.
Project description:ObjectivesNuclear factor-κB is a critical regulator of cell-survival genes and the host inflammatory response. The purpose of this study was to investigate the role of enterocyte-specific NF-kB in sepsis through selective ablation of IkB kinase.DesignProspective, randomized controlled study.SettingAnimal laboratories in university medical centers.Subjects and interventionsMice lacking functional NF-kB in their intestinal epithelium (Vil-Cre/Ikkβ) and wild-type mice were subjected to sham laparotomy or cecal ligation and puncture. Animals were killed at 24 hours or followed 7 days for survival.Measurements and main resultsSeptic wild-type mice had decreased villus length compared with sham mice, whereas villus atrophy was further exacerbated in septic Vil-Cre/Ikkβ mice. Sepsis induced an increase in intestinal epithelial apoptosis compared with sham mice, which was further exacerbated in Vil-Cre/Ikkβ mice. Sepsis induced intestinal hyperpermeability in wild-type mice compared with sham mice, which was further exacerbated in septic Vil-Cre/Ikkβ mice. This was associated with increased intestinal expression of claudin-2 in septic wild-type mice, which was further increased in septic Vil-Cre/Ikkβ mice. Both, pro-inflammatory and anti-inflammatory cytokines were increased in serum following cecal ligation and puncture, and interleukin 10 and monocyte chemoattractant protein-1 levels were higher in septic Vil-Cre/Ikkβ mice than in septic wild-type mice. All septic mice were bacteremic, but no differences in bacterial load were identified between wild-type and Vil-Cre/Ikkβ mice. To determine the functional significance of these results, animals were followed for survival. Septic wild-type mice had lower mortality than septic Vil-Cre/Ikkβ mice (47% vs 80%, p<0.05). Antitumor necrosis factor administration decreased intestinal apoptosis, permeability, and mortality in wild-type septic mice, and a similar improvement in intestinal integrity and survival were seen when antitumor necrosis factor was given to Vil-Cre/Ikkβ mice.ConclusionsEnterocyte-specific NF-kB has a beneficial role in sepsis by partially preventing sepsis-induced increases in apoptosis and permeability, which are associated with worsening mortality.
Project description:Metformin is currently the most prescribed drug for treatment of type 2 diabetes mellitus in humans. It has been well established that long-term treatment with metformin improves glucose tolerance in mice by inhibiting hepatic gluconeogenesis. Interestingly, a single dose of orally administered metformin acutely lowers blood glucose levels, however, little is known about the mechanism involved in this effect. Glucose tolerance, as assessed by the glucose tolerance test, was improved in response to prior oral metformin administration when compared to vehicle-treated mice, irrespective of whether the animals were fed either the standard or high-fat diet. Blood glucose-lowering effects of acutely administered metformin were also observed in mice lacking functional AMP-activated protein kinase, and were independent of glucagon-like-peptide-1 or N-methyl-D-aspartate receptors signaling. [18F]-FDG/PET revealed a slower intestinal transit of labeled glucose after metformin as compared to vehicle administration. Finally, metformin in a dose-dependent but indirect manner decreased glucose transport from the intestinal lumen into the blood, which was observed ex vivo as well as in vivo. Our results support the view that the inhibition of transepithelial glucose transport in the intestine is responsible for lowering blood glucose levels during an early response to oral administration of metformin.
Project description:Oral vaccination is one of the most promising applications of polymeric nanoparticles. Using two different in vitro cellular models to partially reproduce the characteristics of intestinal enterocytes and M-cells, this study demonstrates that nanoparticle transport through the M-cell co-culture model is 5-fold that of the intestinal epithelial monolayer, with at least 80% of the chitosan-DNA nanoparticles uptaken in the first 30 min. Among the properties of nanoparticles studied, ligand decoration has the most dramatic effect on the transcytosis rate: transferrin modification enhances transport through both models by 3- to 5-fold. The stability of the nanoparticles also affects transport kinetics. Factors which de-stabilize the nanoparticles, such as low charge (N/P) ratio and addition of serum, result in aggregation and in turn decreases transport efficiency. Of these stability factors, luminal pH is of great interest as an increase in pH from 5.5 to 6.4 and 7.4 leads to a 3- and 10-fold drop in nanoparticle transport, respectively. Since soluble chitosan can act as an enhancer to increase paracellular transport by up to 60%, this decrease is partially attributed to the soluble chitosan precipitating near neutral pH. The implication that chitosan-DNA nanoparticles are more stable in the upper regions of the small intestine suggests that higher uptake rates may occur in the duodenum compared to the ileum and the colon.
