Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes) where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules, albeit with generally lower affinity than FA. The significance of these lower affinity interactions with exogenous compounds is not known. In this manuscript, we describe further characterization of drug-rat I-FABP binding interactions using a thermal-shift assay. A structural explanation of the observed affinity of rat I-FABP for different drugs based on spectroscopic data and modeling experiments is presented. In addition, immunocytochemistry has been used to probe the expression of I-FABP in a cell culture model reflective of the absorptive cells of the small intestine. Taken together, these data suggest a possible role for I-FABP in the disposition of some lipophilic drugs within the enterocyte.

Examination of the role of intestinal Fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein—intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen {approx} bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

The relationship between monocarboxylate transporters 1 and 4 expression in skeletal muscle and endurance performance in athletes

The purpose of this study was to examine the relationship between skeletal muscle monocarboxylate transporters 1 and 4 (MCT1 and MCT4) expression, skeletal muscle oxidative capacity and endurance performance in trained cyclists. Ten well-trained cyclists (mean ± SD; age 24.4 ± 2.8 years, body mass 73.2 ± 8.3 kg, VO2max 58 ± 7 ml kg−1 min−1) completed three endurance performance tasks [incremental exercise test to exhaustion, 2 and 10 min time trial (TT)]. In addition, a muscle biopsy sample from the vastus lateralis muscle was analysed for MCT1 and MCT4 expression levels together with the activity of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD). There was a tendency for VO2max and peak power output obtained in the incremental exercise test to be correlated with MCT1 (r = −0.71 to −0.74; P < 0.06), but not MCT4. The average power output (P average) in the 2 min TT was significantly correlated with MCT4 (r = −0.74; P < 0.05) and HAD (r = −0.92; P < 0.01). The P average in the 10 min TT was only correlated with CS activity (r = 0.68; P < 0.05). These results indicate the relationship between MCT1 and MCT4 as well as cycle TT performance may be influenced by the length and intensity of the task.

The effect of vitamin : a deficiency on glucose uptake in the rat

This thesis describes an investigation of the effects of vitamin A deficiency on gut function, The central hypothesis to be tested was that acute vitamin A deficiency affects glucose uptake from the small intestine- The hypothesis was tested using a system involving perfusion of isolated segments of the small intestine in the anaesthetized rat. The system was used to study effects on glucose uptake under steady-state conditions. In the initial part of the study, experiments were diverted towards setting up the system for measuring steady-state uptake, and determining the relative contributions of active uptake and diffusion. Phenol red was found to be a reliable non-absorbable marker for determining net water movement. Phlorizin, generally at 1 mmol/L, was used as a competitive (reversible) inhibitor of active uptake. It is difficult however to confirm complete inhibition of active uptake by phlorizin because of the limited solubility of the inhibitor. The kinetics of glucose uptake f ram intra-luminal maltose were found to be, in general, not significantly different from those applying to the uptake of glucose from an equivalent glucose solution. Maltase activity in the perfused gut segment was found to be sufficient to hydrolyse most of the maltose (80 per cent or more) in the solution being perfused, a much greater proportion than was absorbed. Glucose absorptive capacity, measured on an intestinal dry weight basis, was greatest in the duodenum and progressively less in the jejunum and ileum. The rate of water uptake f ran the gut was increased by the presence of glucose in the lumen, and was linked to glucose uptake as shown by the inhibition of water uptake by phlorizin. Uptake of glucose by solvent drag was demonstrated by showing an increased rate of glucose uptake when the rate of water uptake was increased by perfusing a solution of reduced osmotic pressure. In the experiment a low intra-luminal glucose concentration was used to preclude net uptake by diffusion and active uptake was blocked with phlorizin. This process was further investigated using streptozotocin-diabetic rats in which the diabetes establishes a hyperosomotic blood with hyperglycaemia. Uptake by solvent drag was more obvious in diabetic animals. A back-diffusion (exsorption) of glucose from the tissues to the lumen was also shown; the rate being proportional to plasma glucose concentration. Vitamin A deficiency was established in weanling rats after 6-7 weeks feeding on a diet based on wheat starch, coconut oil, and casein washed with hot ethanol, together with vitamins and minerals. The vitamin A deficiency led to classic eye signs and was reversed by the addition to the diet of retinoic acid (5 g/g diet). Vitamin A deficiency decreased intestinal mucus production (dry weight) but had no detectable effect on the histology of the villous epithelium as shown under the light microscope. Using perfusion experiments it was shown that vitamin A deficiency had no significant effect on the rate of active uptake of glucose, but that deficiency increased the rate of passive uptake.

Interaction of dietary fibre, carbohydrate metabolism and diabetes in the rat

It is currently accepted that the most appropriate diet in the treatment of non-insulin-dependent diabetes mellitus "e;NIDDM"e; is high in carbohydrates, high in fibre and low in fat. Dietary fibre reduces the rate of carbohydrate absorption, which may have a beneficial effect on insulin action. Furthermore, high fibre diets also increase the amount of carbohydrates which are not absorbed from the small intestine. These malabsorbed carbohydrates are fermented by the bacterial population in the large intestine, producing short chain fatty acids "e;SCFA"e;, including propionate, which has been shown to alter liver carbohydrate metabolism. This thesis investigated the actions of slowed carbohydrate absorption and carbohydrate malabsorption in streptozotocin-induced "e;STZ"e; diabetic rats. High carbohydrate diet supplemented with guar gum, a soluble dietary fibre, fed to STZ diabetic rats improved insulin sensitivity. investigation of the alterations in the stomach and small intestine demonstrated that guar increased the viscosity of the meal in the intestine. The action of increased fermentation, producing more propionate, was investigated by supplementing propionate into the diets of STZ diabetic rats or when perfused into isolated rat livers. No changes in insulin action or liver glucose metabolism were measured. in addition, it was shown that guar gum reduces food intake in STZ diabetic rats. Mild reductions in food intake in STZ diabetic rats were shown to increase insulin action. In summary, STZ diabetic rats fed high carbohydrate, high fibre diets reductions in food consumption and slowed carbohydrate absorption are important factors which may lower blood glucose concentrations and increase insulin action. increased SCFA production is unlikely to contribute significantly to the improvements in insulin action.

Adrenergic regulation of carbohydrate metabolism during exercise

1. This series of studies was undertaken to examine the adrenergic regulation of carbohydrate metabolism during exercise. Recreationally active males were tested during moderate to intense exercise on a stationary cycle ergometer. Venous and arterial plasma obtained from indwelling catheters was analysed for hormonal and metabolite responses, and hepatic glucose production and glucose uptake were measured using the tracer-dilution method with stable isotopes. Muscle samples were obtained by the needle biopsy technique to examine muscle glycogen utilisation and the flux of related muscle metabolites using enzymatic, fluorometric and radioisotopic techniques. 2. During moderate exercise adrenaline infusion induced a marked hyperglycemia and this was due to reduced glucose uptake rather than enhanced hepatic glucose production. The reduction in glucose uptake was most likely mediated by a decrease in glucose phosphorylation, as indicated by the accumulation of glucose 6-phosphate with adrenaline infusion. 3. The hyperglycemic response to intense exercise was prevented by the administration of α- and β-adrenergic antagonists. Adrenergic blockade was without effect on hepatic glucose production whereas glucose uptake was enhanced when compared with control subjects. These data support the notion that adrenergic mechanisms are more important in restraining glucose uptake than enhancing hepatic glucose production during intense exercise. Other glucoregulatory factors are responsible for the increase in glucose production during intense exercise. 4. Elevated plasma adrenaline levels during moderate exercise in untrained men increases skeletal muscle glycogen breakdown and PDH activation which results

Reduced glycogen availability is associated with increased AMPKα2 activity, nuclear AMPKα2 protein abundance, and GLUT4 mRNA expression in contracting human skeletal muscle

Glycogen availability can influence glucose transporter 4 (GLUT4) expression in skeletal muscle through unknown mechanisms. The multisubstrate enzyme AMP-activated protein kinase (AMPK) has also been shown to play an important role in the regulation of GLUT4 expression in skeletal muscle. During contraction, AMPK [alpha]2 translocates to the nucleus and the activity of this AMPK isoform is enhanced when skeletal muscle glycogen is low. In this study, we investigated if decreased pre-exercise muscle glycogen levels and increased AMPK [alpha]2 activity reduced the association of AMPK with glycogen and increased AMPK [alpha]2 translocation to the nucleus and GLUT4 mRNA expression following exercise. Seven males performed 60 min of exercise at ~70% [VO.sub.2] peak on 2 occasions: either with normal (control) or low (LG) carbohydrate pre-exercise muscle glycogen content. Muscle samples were obtained by needle biopsy before and after exercise. Low muscle glycogen was associated with elevated AMPK [alpha]2 activity and acetyl-CoA carboxylase [beta] phosphorylation, increased translocation of AMPK [alpha]2 to the nucleus, and increased GLUT4 mRNA. Transfection of primary human myotubes with a constitutively active AMPK adenovirus also stimulated GLUT4 mRNA, providing direct evidence of a role of AMPK in regulating GLUT4 expression. We suggest that increased activation of AMPK [alpha]2 under conditions of low muscle glycogen enhances AMPK [alpha]2 nuclear translocation and increases GLUT4 mRNA expression in response to exercise in human skeletal muscle.

Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients

Aims/hypothesis: The 5′-AMP-activated protein kinase (AMPK) pathway is intact in type 2 diabetic patients and is seen as a target for diabetes treatment. In this study, we aimed to assess the impact of the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) on both glucose and fatty acid metabolism in vivo in type 2 diabetic patients.

Methods: Stable isotope methodology and blood and muscle biopsy sampling were applied to assess blood glucose and fatty acid kinetics following continuous i.v. infusion of AICAR (0.75 mg kg−1 min−1) and/or NaCl (0.9%) in ten male type 2 diabetic patients (age 64 ± 2 years; BMI 28 ± 1 kg/m2).
Results Plasma glucose rate of appearance (R a) was reduced following AICAR administration, while plasma glucose rate of disappearance (R d) was similar in the AICAR and control test. Consequently, blood glucose disposal (R d expressed as a percentage of R a) was increased following AICAR infusion (p < 0.001). Accordingly, a greater decline in plasma glucose concentration was observed following AICAR infusion (p < 0.001). Plasma NEFA R a and R d were both significantly reduced in response to AICAR infusion, and were accompanied by a significant decline in plasma NEFA concentration. Although AMPK phosphorylation in skeletal muscle was not increased, we observed a significant increase in acetyl-CoA carboxylase phosphorylation (p < 0.001).

Conclusions/interpretation: The i.v. administration of AICAR reduces hepatic glucose output, thereby lowering blood glucose concentrations in vivo in type 2 diabetic patients. Furthermore, AICAR administration stimulates hepatic fatty acid oxidation and/or inhibits whole body lipolysis, thereby reducing plasma NEFA concentration.

Bioassay detects soluble MAdCAM-1 in body fluids

Mucosal addressin cell adhesion molecule (MAdCAM-1) is a key player in mediating the infiltration of leucocytes into chronically inflamed tissues. Five anti-MAdCAM-1 monoclonal antibodies (mAb), designated 17F5, 201F7, 314G8, 377D10 and 355G8, were generated by fusion of P3 × 63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with recombinant human MAdCAM-1-Fc. The latter four mAb recognize the ligand-binding first Ig domain, and block T -cell adhesion to MAdCAM-1. The non-blocking mAb 17F5 recognizes the mucin domain. Extensive analysis of a large panel of paraffin-embedded human tissues revealed that the 314G8 mAb detected MAdCAM-1 on venules in the spleen and small intestine. MAdCAM-1 was strongly expressed in the synovium of osteoarthritis patients, predominantly on the endothelial lining of blood vessels, but also within the vessel lumen. An ELISA, based on mAb 377D10 and 355G8, was developed to determine whether soluble MAdCAM-1 was present in body fluids, and to measure the levels present. The assay detected soluble MAdCAM-1 in the serum and urine of healthy donors, at levels similar to those of soluble forms of the related CAM, ICAM-1 and VCAM-1. The anti-MAdCAM-1 antibodies and assay developed here may be useful therapeutically in the treatment of inflammation in humans. Similarly, they may be useful diagnostically to monitor the presence and levels of MAdCAM-1.

Some osmoregulatory parameters in selected fishes of the Australasian region

Fish must maintain their internal ionic concentration. This occurs through co-ordination of cellular ion channels and transporters, and hormones acting on and within the cells of the gill, kidney, intestine, and, in some species, unique organs. This study measured some of these transporters and hormones in unusual species of the Australasian region.

The role of copper transport proteins in the small intestines

Physiological copper homeostasis involves striking a balance between absorption and secretion. ATP7A was identified at the trans-Golgi network but relocalized to vesicles under copper exposure in the intestine. This suggests that ATP7A may be a rate limiting step in intestinal uptake of copper.

Haptically simulating soft tissue using spherical voxel and reactive agents

This thesis describes technology developed by the author enabling trainee surgeons to perform needle insertion procedures with force feedback (haptics) on a virtual patient. Addition of the sense of touch to medical simulation is arguably the most important step forward in the evolution of haptic technology to this day.

The case | Allograft dysfunction in a patient with sickle cell disease

The article focuses on the study concerning the diagnosis of acute renal allograft in patient with sickle cell disease. The 33 year old underwent several surgeries with immediate graft function. The percutaneous renal biopsy of the patient shows focal cortical necrosis and acute tubular damage coherent with ischemia. It concludes that the allograft sickling is the primary cause of the acute renal dysfunction in patients with sickle cell disease.

An appraisal of the histopathological assessment of liver fibrosis.

One of the most important aspects of the histopathological assessment of liver biopsies in the setting of chronic liver disease is determination of the degree of fibrosis and architectural change. Most of the work in this regard has been concerned with chronic viral hepatitis. This article attempts to assess critically our current and historical biopsy practice, from subjective fibrosis scoring systems to biopsy sample size; and the appropriate use of the data that scoring systems
generate in the research and clinical setting. An understanding of the limitations of each of the components of the fibrosis assessment process can help to devise appropriate protocols to ensure that the information obtained is optimised, and its degree of reliability appreciated. It is only from this starting point that recently promulgated antifibrotic medications and ‘‘non-invasive’’ liver fibrosis assessment techniques can be evaluated properly.