Characterization of the drug binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in the cytosolic solubilization of fatty acids during fat absorption. In the current studies, the interaction of L-FABP with a range of lipophilic drugs has been evaluated to explore the potential for L-FABP to provide an analogous function during the absorption of lipophilic drugs. Binding affinity for L-FABP was assessed by displacement of a fluorescent marker, 1-anilinonaphthalene-8-sulfonic acid (ANS), and the binding site location was determined via nuclear magnetic resonance chemical shift perturbation studies. It was found that the majority of drugs bound to L-FABP at two sites, with the internal site generally having a higher affinity for the compounds tested. Furthermore, in contrast to the interaction of L-FABP with fatty acids, it was demonstrated that a terminal carboxylate is not required for specific binding of lipophilic drugs at the internal site of L-FABP.

Thermodynamics of lipophilic drug binding to intestinal fatty acid binding protein and permeation across membranes

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. Thus, in addition to its central role in FA trafficking, I-FABP potentially serves as an important intracellular carrier of lipophilic drugs. In this study we provide a detailed thermodynamic analysis of the binding and stability properties of I-FABP in complex with a series of fibrate and fenamate drugs to provide an insight into the forces driving drug binding to I-FABP. Drug binding and selectivity for I-FABP are driven by the interplay of protein−ligand interactions and solvent processes. The Gibbs free energies (ΔG°) determined from dissociation constants at 25 °C ranged from −6.2 to −10 kcal/mol. The reaction energetics indicate that drug binding to I-FABP is an enthalpy−entropy driven process. The relationship between I-FABP stability and drug binding affinity was examined by pulse proteolysis. There is a strong coupling between drug binding and I-FABP stability. The effect of an I-FABP protein sink on the kinetics and thermodynamics of tolfenamic acid permeation across an artificial phospholipid membrane were investigated. I-FABP significantly decreased the energy barrier for desorption of tolfenamic acid from the membrane into the acceptor compartment. Taken together, these data suggest that the formation of stable drug−I-FABP complexes is thermodynamically viable under conditions simulating the reactant concentrations likely observed in vivo and maybe a significant biochemical process that serves as a driving force for passive intestinal absorption of lipophilic drugs.

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.

Probing the fibrate binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

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.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

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.

A protocol for the combined sub-fractionation and delipidation of lipid-binding proteins by hydrophobic interaction chromatography

Cellular lipids frequently co-purify with lipid binding proteins isolated from tissue extracts or heterologous host systems and as such hinder in vitro ligand binding approaches for which the apo-protein is a prerequisite. Here we present a technique for the complete removal of unesterified fatty acids, phospholipids, steroids and other lipophilic ligands bound to soluble proteins, without protein denaturation. Peroxisome proliferator activated receptor gamma ligand binding domain and intracellular fatty acid binding proteins were expressed in an Escherichia coli host and completely delipidated by hydrophobic interaction chromatography using phenyl sepharose. The delipidation procedure operates at room temperature with complete removal of bound lipids in a single step, as ascertained by mass spectrometry analysis of organic solvent extracts from purified protein samples. The speed and capacity of this method makes it amenable to scale-up and high-throughput applications. The method can also easily be adapted for other lipid binding proteins that require delipidation under native conditions.

A short-term, high-fat diet up-regulates lipid metabolism and gene expression in human skeletal muscle

Background: Dietary fatty acids may be important in regulating gene expression. However, little is known about the effect of changes in dietary fatty acids on gene regulation in human skeletal muscle.
Objective: The objective was to determine the effect of altered dietary fat intake on the expression of genes encoding proteins necessary for fatty acid transport and ß-oxidation in skeletal muscle.
Design: Fourteen well-trained male cyclists and triathletes with a mean (± SE) age of 26.9 ± 1.7 y, weight of 73.7 ± 1.7 kg, and peak oxygen uptake of 67.0 ± 1.3 mL ˙ kg-1 ˙ min-1 consumed either a high-fat diet (HFat: > 65% of energy as lipids) or an isoenergetic high-carbohydrate diet (HCho: 70–75% of energy as carbohydrate) for 5 d in a crossover design. On day 1 (baseline) and again after 5 d of dietary intervention, resting muscle and blood samples were taken. Muscle samples were analyzed for gene expression [fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), carnitine palmitoyltransferase I (CPT I), ß-hydroxyacyl-CoA dehydrogenase (ß-HAD), and uncoupling protein 3 (UCP3)] and concentrations of the proteins FAT/CD36 and FABPpm.
Results: The gene expression of FAT/CD36 and &szlig; -HAD and the gene abundance of FAT/CD36 were greater after the HFat than after the HCho diet (P < 0.05). Messenger RNA expression of FABPpm, CPT I, and UCP-3 did not change significantly with either diet.
Conclusions: A rapid and marked capacity for changes in dietary fatty acid availability to modulate the expression of mRNA-encoding proteins is necessary for fatty acid transport and oxidative metabolism. This finding is evidence of nutrient-gene interactions in human skeletal muscle.

The effect of cocoa supplementation on hepatic steatosis, reactive oxygen species and LFABP in a rat model of NASH

Background: Non alcoholic steatohepatitis is hypothesised to develop via a mechanism involving fat accumulation and oxidative stress. The current study aimed to investigate if an increase in oxidative stress was associated with changes in the expression of liver fatty acid binding protein in a rat model of non alcoholic steatohepatitis and whether cocoa supplementation attenuated those changes.

Methods: Female Sprague Dawley rats were fed a high fat control diet, a high fat methionine choline deficient diet, or one of four 12.5% cocoa supplementation regimes in combination with the high fat methionine choline deficient diet.

Results: Liver fatty acid binding protein mRNA and protein levels were reduced in the liver of animals with fatty liver disease when compared to controls. Increased hepatic fat content was accompanied by higher levels of oxidative stress in animals with fatty liver disease when compared to controls. An inverse association was found between the levels of hepatic liver fatty acid binding protein and the level of hepatic oxidative stress in fatty liver disease. Elevated NADPH oxidase protein levels were detected in the liver of animals with increased severity in inflammation and fibrosis. Cocoa supplementation was associated with partial attenuation of these pathological changes, although the severity of liver disease induced by the methionine choline deficient diet prevented complete reversal of any disease associated changes. Red blood cell glutathione was increased by cocoa supplementation, whereas liver glutathione was reduced by cocoa compared to methionine choline deficient diet fed animals.

Conclusion: These findings suggest a potential role for liver fatty acid binding protein and NADPH oxidase in the development of non alcoholic steatohepatitis. Furthermore, cocoa supplementation may have be of therapeutic benefit in less sever forms of NASH.

Dietary protein level interacts with ω-3 polyunsaturated fatty acid deficiency to induce hypertension

Background : Dietary ω-3 fatty acid deficiency can lead to hypertension in later life; however, hypertension is affected by numerous other dietary factors. We examined the effect of altering the dietary protein level on blood pressure in animals deficient or sufficient in ω-3 fatty acids.

Methods : Female rats were placed on one of four experimental diets 1 week prior to mating. Diets were either deficient (10% safflower oil; DEF) or sufficient (7% safflower oil, 3% flaxseed oil; SUF) in ω-3 fatty acids and contained 20 or 30% casein (DEF20, SUF20, DEF30, SUF30). Offspring were maintained on the maternal diet for the duration of the experiment. At 12, 18, 24, and 30 weeks, blood pressure was assessed by tail cuff plethysmography.

Results : At both 12 and 18 weeks of age, no differences in blood pressure were observed based on diet, however, by 24 weeks hypertension was evident in DEF30 animals; there were no blood pressure differences between the other groups. This hypertension in DEF30 group was increased at 30 weeks, with systolic, diastolic, and mean arterial pressure all elevated.

Conclusions : These results indicate that the hypertension previously attributed to ω-3 fatty acid deficiency is dependent on additional dietary factors, including protein content. Furthermore, this study is the first to plot the establishment of ω-3 fatty acid deficiency hypertension over time.

Lipid and fatty acid digestibility of three oil types in the Australian shortfin eel, Anguilla australis

The lipid and fatty acid digestibilities of three semi-purified, isonitrogenous (48.9–50.8% protein) and isocalorific (19.1–20.8 kJ g⁻¹) diets, in which the lipid source was either cod liver oil (CLO), linseed oil (LO) or sunflower oil (SFO), were estimated in the Australian shortfin eel (Anguilla australis) using chromic oxide as an external marker. Apparent percent protein and energy digestibilities of the diets were not significantly (P>0.05) affected by the lipid source, but the lipid digestibility was. The percent apparent lipid digestibility was lowest in the LO diet (90.2±0.6) and highest in the CLO diet (95.6±0.2).

Not all the fatty acids present in any one diet were recovered in the faecal samples. In diets with CLO, only three saturates (out of five), five monoenes and six (out of 11) PUFAs were detected in faecal samples. With all the diets, 20:0 and 22:0, and none of the n−6 HUFA were detected in the faecal samples. The digestibility of all the fatty acids, except 18:3n−3, was lowest in the diet with LO, and significantly so (P>0.05) from the other diets.

In shortfin eel, there was a trend for the digestibility of saturated fatty acids of diets with the animal oil as the lipid source to decrease with increasing chain length, and in diets with vegetable oil to increase initially and then decrease. A somewhat comparable trend was also evident in respect of monoenes.

When the digestibility of different categories of fatty acids is considered, the digestibility of saturates, monoenes, unsaturates, n−6, PUFA, HUFA and total fatty acid digestibilities of LO diet were the lowest, and differed significantly (P<0.05) from those of the CLO and SFO diets, except in the case of n−3 fatty acids.

Effects of dietary oil source on growth and fillet fatty Acid composition of Murray cod, Maccullochella peelii peelii

The Murray cod, an Australian native freshwater fish, supports a relatively small but increasing aquaculture industry in Australia. Presently, there are no dedicated commercial diets available for Murray cod; instead, nutritionally sub-standard feeds formulated for other species are commonly used. The aim of the present investigation was to assess the suitability of two plant based lipid sources, canola oil (CO) and linseed oil (LO), as alternatives to fish oil for juvenile Murray cod. Five iso-nitrogenous, iso-calorific, iso-lipidic semi-purified experimental diets were formulated with 17% lipid originating from 100% cod liver oil (FO), 100% canola oil, 100% linseed oil and 1 : 1 blends of canola and cod liver oil (CFO) and 1 : 1 blends of linseed and cod liver oil (LFO). Each of the diets was fed to apparent satiation twice daily to triplicate groups of 50 Murray cod with initial mean weights of 6.45 ± 1.59 g for 84 days at 22 °C. Final mean weight, specific growth rate and daily feed consumption were significantly higher for the FO and LFO treatments compared to the LO treatment. Feed conversion and protein efficiency ratios were not significantly different amongst treatments. Experimental diets containing vegetable oil and vegetable oil blend(s) had significantly higher concentrations of n-6 fatty acids, predominantly in the form of linoleic acid (LA), while n-3 fatty acids were present in significantly higher concentrations in LO and LFO treatments. The fatty acid composition of Murray cod fillet was reflective of the dietary lipid source. Fillet of fish fed the FO was highest in EPA (20:5n-3), ArA (20:4n-6) and DHA (22:6n-3). Fish fed the CO diet had high concentrations of oleic acid (OlA) (192.2 ± 10.5 mg g lipid− 1), while the fillet of Murray cod fed the LO diet was high in α-linolenic acid (LnA) (107.1 ± 6.7 mg g lipid− 1). The present study suggests that fish oil can be replaced by up to 100% with canola oil and by up to 50% with linseed oil in Murray cod diets with no significant effect on growth.

Impaired activation of AMP-Kinase and fatty acid oxidation by globular adiponectin in cultured human skeletal muscle of obese type 2 diabetics

Adiponectin is an adipocyte-derived hormone associated with antidiabetic actions. In rodent skeletal muscle, globular adiponectin (gAD) activates AMP-kinase (AMPK) and stimulates fatty acid oxidation effects mediated through the adiponectin receptors, AdipoR1 and AdipoR2. In the present study, we examined the mRNA expression of adiponectin receptors and the effects of gAD on AMPK activity and fatty acid oxidation in skeletal muscle myotubes from lean, obese, and obese type 2 diabetic subjects. Myotubes from all groups expressed approximately 4.5-fold more AdipoR1 mRNA than AdipoR2, and obese subjects tended to have higher AdipoR1 expression (P = 0.052). In lean myotubes, gAD activates AMPK[alpha]1 and -[alpha]2 by increasing Thr172 phosphorylation, an effect associated with increased acetyl-coenzyme A carboxylase (ACC[beta]) Ser221 phosphorylation and enhanced rates of fatty acid oxidation, effects similar to those observed after pharmacological AMPK activation by 5-aminoimidazole-4-carboxamide riboside. In obese myotubes, the activation of AMPK signaling by gAD at low concentrations (0.1 [mu]g/ml) was blunted, but higher concentrations (0.5 [mu]g/ml) stimulated AMPK[alpha]1 and -[alpha]2 activities, AMPK and ACC[beta] phosphorylation, and fatty acid oxidation. In obese type 2 diabetic myotubes, high concentrations of gAD stimulated AMPK[alpha]1 activity and AMPK phosphorylation; however, ACC[beta] phosphorylation and fatty acid oxidation were unaffected. Reduced activation of AMPK signaling and fatty acid oxidation in obese and obese diabetic myotubes was not associated with reduced protein expression of AMPK[alpha] and ACC[beta] or the expression and activity of the upstream AMPK kinase, LKB1. These data suggest that reduced activation of AMPK by gAD in obese and obese type 2 diabetic subjects is not caused by reduced adiponectin receptor expression but that aspects downstream of the receptor may inhibit AMPK signaling.