A novel human tau knock-in mouse model reveals interaction of Abeta and human tau under progressing cerebral amyloidosis in 5xFAD mice.

BACKGROUND: Hyperphosphorylation and intraneuronal aggregation of the microtubule-associated protein tau is a major pathological hallmark of Alzheimer's disease (AD) brain. Of special interest is the effect of cerebral amyloid beta deposition, the second main hallmark of AD, on human tau pathology. Therefore, studying the influence of cerebral amyloidosis on human tau in a novel human tau knock-in (htau-KI) mouse model could help to reveal new details on their interplay. METHODS: We studied the effects of a novel human htau-KI under fast-progressing amyloidosis in 5xFAD mice in terms of correlation of gene expression data with human brain regions, development of Alzheimer's-like pathology, synaptic transmission, and behavior. RESULTS: The main findings are an interaction of human beta-amyloid and human tau in crossbred 5xFADxhtau-KI observed at transcriptional level and corroborated by electrophysiology and histopathology. The comparison of gene expression data of the 5xFADxhtau-KI mouse model to 5xFAD, control mice and to human AD patients revealed conspicuous changes in pathways related to mitochondria biology, extracellular matrix, and immune function. These changes were accompanied by plaque-associated MC1-positive pathological tau that required the htau-KI background. LTP deficits were noted in 5xFAD and htau-KI mice in contrast to signs of rescue in 5xFADxhtau-KI mice. Increased frequencies of miniature EPSCs and miniature IPSCs indicated an upregulated presynaptic function in 5xFADxhtau-KI. CONCLUSION: In summary, the multiple interactions observed between knocked-in human tau and the 5xFAD-driven progressing amyloidosis have important implications for future model development in AD.

Synthesis and structure-activity relationships of pyrazole-based inhibitors of meprin α and ß.

Targeting metalloproteinases has been in the focus of drug design for a long time. However, meprin α and ß emerged as potential drug targets just recently and are linked to several diseases with different pathological background. Nevertheless, the validation of meprins as suitable drug targets still requires highly potent and selective inhibitors as chemical probes to elucidate their role in pathophysiology. Albeit highly selective inhibitors of meprin ß have already been reported, only inhibitors of meprin α with modest activity or selectivity are known. Starting from recently reported heteroaromatic scaffolds, the aim of this study was the optimisation of meprin α and/or meprin ß inhibition while keeping the favourable off-target inhibition profile over other metalloproteases. We report potent pan-meprin inhibitors as well as highly active inhibitors of meprin α with superior selectivity over meprin ß. The latter are suitable to serve as chemical probes and enable further target validation.

Evidence for Enhanced Efficacy of Passive Immunotherapy against Beta-Amyloid in CD33-Negative 5xFAD Mice.

Passive immunotherapy is a very promising approach for the treatment of Alzheimer's disease (AD). Among the different antibodies under development, those targeting post-translationally modified Aß peptides might combine efficient reduction in beta-amyloid accompanied by lower sequestration in peripheral compartments and thus anticipated and reduced treatment-related side effects. In that regard, we recently demonstrated that the antibody-mediated targeting of isoD7-modified Aß peptides leads to the attenuation of AD-like amyloid pathology in 5xFAD mice. In order to assess novel strategies to enhance the efficacy of passive vaccination approaches, we investigated the role of CD33 for Aß phagocytosis in transgenic mice treated with an isoD7-Aß antibody. We crossbred 5xFAD transgenic mice with CD33 knock out (CD33KO) mice and compared the amyloid pathology in the different genotypes of the crossbreds. The knockout of CD33 in 5xFAD mice leads to a significant reduction in Aß plaques and concomitant rescue of behavioral deficits. Passive immunotherapy of 5xFAD/CD33KO showed a significant increase in plaque-surrounding microglia compared to 5xFAD treated with the antibody. Additionally, we observed a stronger lowering of Aß plaque load after passive immunotherapy in 5xFAD/CD33KO mice. The data suggest an additive effect of passive immunotherapy and CD33KO in terms of lowering Aß pathology. Hence, a combination of CD33 antagonists and monoclonal antibodies might represent a strategy to enhance efficacy of passive immunotherapy in AD.

Heteroaromatic Inhibitors of the Astacin Proteinases Meprin α, Meprin ß and Ovastacin Discovered by a Scaffold-Hopping Approach.

Astacin metalloproteinases, in particular meprins α and ß, as well as ovastacin, are emerging drug targets. Drug-discovery efforts have led to the development of the first potent and selective inhibitors in the last few years. However, the most recent compounds are based on a highly flexible tertiary amine scaffold that could cause metabolic liabilities or decreased potency due to the entropic penalty upon binding to the target. Thus, the aim of this study was to discover novel conformationally constrained scaffolds as starting points for further inhibitor optimization. Shifting from flexible tertiary amines to rigid heteroaromatic cores resulted in a boost in inhibitory activity. Moreover, some compounds already exhibited higher activity against individual astacin proteinases compared to recently reported inhibitors and also a favorable off-target selectivity profile, thus qualifying them as very suitable chemical probes for target validation.

Targeting isoaspartate-modified Aß rescues behavioral deficits in transgenic mice with Alzheimer's disease-like pathology.

BACKGROUND: Amyloid ß (Aß)-directed immunotherapy has shown promising results in preclinical and early clinical Alzheimer's disease (AD) trials, but successful translation to late clinics has failed so far. Compelling evidence suggests that post-translationally modified Aß peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such Aß variants have been initiated. Modified Aß represents a small fraction of deposited material in plaques compared to pan-Aß epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize L-isoaspartate-modified Aß (isoD7-Aß) and tested a lead antibody molecule in 5xFAD mice. METHODS: This work comprises a combination of chemical and biochemical techniques as well as behavioral analyses. Aß peptides, containing L-isoaspartate at position 7, were chemically synthesized and used for immunization of mice and antibody screening methods. Biochemical methods included anti-isoD7-Aß monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-Aß ELISA as well as different non-modified Aß ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-Aß antibody K11_IgG2a was applied intraperitoneally to 5xFAD mice for 38 weeks. Treatment controls implemented were IgG2a isotype as negative and 3D6_IgG2a, the parent molecule of bapineuzumab, as positive control antibodies. Behavioral studies included elevated plus maze, pole test, and Morris water maze. RESULTS: Our advanced antibody K11 showed a KD in the low nM range and > 400fold selectivity for isoD7-Aß compared to other Aß variants. By using this antibody, we demonstrated that formation of isoD7-Aß may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged Aß from newly formed peptides. Importantly, we also show that the Tottori mutation responsible for early-onset AD in a Japanese pedigree is characterized by massively accelerated formation of isoD7-Aß in cell culture. The presence of isoD7-Aß was verified by K11 in post mortem human cortex and 5xFAD mouse brain tissue. Passive immunization of 5xFAD mice resulted in a significant reduction of isoD7-Aß and total Aß in brain. Amelioration of cognitive impairment was demonstrated by Morris water maze, elevated plus maze, pole, and contextual fear conditioning tests. Interestingly, despite the lower abundance of the isoD7-Aß epitope, the application of anti-isoD7-Aß antibodies showed comparable treatment efficacy in terms of reduction of brain amyloid and spatial learning but did not result in an increase of plasma Aß concentration as observed with 3D6 treatment. CONCLUSIONS: The present study demonstrates, for the first time, that the antibody-mediated targeting of isoD7-modified Aß peptides leads to attenuation of AD-like amyloid pathology. In conjunction with previously published data on antibodies directed against pGlu-modified Aß, the results highlight the crucial role of modified Aß peptides in AD pathophysiology. Hence, the results also underscore the therapeutic potential of targeting modified amyloid species for defining tailored approaches in AD therapy.

Viromers as carriers for mRNA-mediated expression of therapeutic molecules under inflammatory conditions.

Therapeutic mRNA delivery has been described for several treatment options, such as vaccination and cancer immunotherapy. However, mRNA delivery has to be accompanied by the development and testing of suitable carrier materials due to the instability of mRNAs in human body fluids. In the present study, we investigated the ability of recently developed Viromers to deliver mRNAs in a classical inflammatory setting. We tested mRNAs coding for active components of preclinical (7ND) and approved (sTNF-RII) biologics, in vitro and in vivo. 7ND is an established blocker of the CCR2 axis, whereas sTNF-RII is the active component of the approved drug Etanercept. Viromer/mRNA complexes were transfected into murine macrophages in vitro. Protein expression was analysed using Luciferase reporter expression and mainly identified in spleen, blood and bone marrow in vivo. 7ND-mRNA delivery led to efficient blockage of monocytes infiltration in thioglycolate-induced peritonitis in mice, underlining the ability of Viromers to deliver a therapeutic mRNA cargo without overt toxicity. Therefore, we propose Viromer-based mRNA delivery as a suitable option for the treatment of inflammatory disorders beyond infusion of biological molecules.

Novel reverse thia-analogs of fosmidomycin: Synthesis and antiplasmodial activity.

Thia analogs of fosmidomycin are potent inhibitors of the non-mevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers displayed antiplasmodial in vitro activity in the low nanomolar range, without apparent cytotoxic effects in HeLa cells. The (S)-(+)-enantiomer of a typical representative selectively inhibited IspC and the growth of P. falciparum in continuous culture. The inhibitor was stable at pH 7.6 and room temperature, and no racemization was observed under these conditions during a period of up to two days. Oxidation of selected thioethers to sulfones reduced antiplasmodial activity and the inhibitory activity against Escherichia coli, Mycobacterium tuberculosis and P. falciparum IspC orthologs.

Structure-Guided Design, Synthesis, and Characterization of Next-Generation Meprin ß Inhibitors.

The metalloproteinase meprin ß emerged as a current drug target for the treatment of a number of disorders, among those fibrosis, inflammatory bowel disease and Morbus Alzheimer. A major obstacle in the development of metalloprotease inhibitors is target selectivity to avoid side effects by blocking related enzymes with physiological functions. Here, we describe the structure-guided design of a novel series of compounds, based on previously reported highly active meprin ß inhibitors. The bioisosteric replacement of the sulfonamide scaffold gave rise to a next generation of meprin inhibitors. Selected compounds based on this novel amine scaffold exhibit high activity against meprin ß and also remarkable selectivity over related metalloproteases, i.e., matrix metalloproteases and A disintegrin and metalloproteinases.

Oral intake of 7-dehydrocholesterol increases vitamin D3 concentrations in the liver and kidney.

INTRODUCTION: Due to the high prevalence of vitamin D deficiency, strategies are needed to improve vitamin D status. Food components can affect vitamin D metabolism and have to be considered when estimating the efficacy of vitamin D supplements. 7-dehydrocholesterol (7-DHC) occurs naturally in food, but its impact on vitamin D metabolism has not yet been examined. METHODS: Three groups of male C57BL/6 mice (n=12 per group) were placed on a diet that contained 0, 2.5 or 5mg 7-DHC per kg diet over a period of 6 weeks. Vitamin D and other sterols in the serum, skin, liver and kidney were quantified by LC-MS/MS. The relative mRNA abundance of hepatic genes encoding vitamin D hydroxylation enzymes and transporters was analyzed by real-time RT-PCR. RESULTS: We found a substantial dose-dependent increase of non-hydroxylated vitamin D3 in the liver and kidney of mice fed a diet containing 7-DHC. The vitamin D3 content in the liver was 2.80±0.61pmol/g, 7.34±4.28pmol/g and 12.9±3.58pmol/g in groups that received 0, 2.5 and 5mg/kg 7-DHC, respectively. In the kidney, the vitamin D3 content of these groups was 1.78±1.17pmol/g, 3.55±1.06 and 6.36±2.29pmol/g, respectively. The serum and tissue concentrations of 25-hydroxyvitamin D3 (25(OH)D3) remained unaffected by 7-DHC. The relative mRNA data provided no plausible mechanism for the observed effects of 7-DHC on vitamin D3. All groups of mice had similar concentrations of cholesterol, desmosterol and 7-DHC in their serum and tissues. CONCLUSION: The current findings provide the first evidence that dietary 7-DHC seems to affect vitamin D metabolism. The underlying mechanism remains elusive and needs further investigation.

Additive effects of lupin protein and phytic acid on aortic calcification in ApoE deficient mice.

Lupin proteins have repeatedly been shown to exhibit lipid lowering properties and reduce aortic calcification in atherosclerosis models. Despite many efforts on its identification, the component which is responsible for the observed effects is still under debate. Phytic acid which is generally associated with lupin protein isolates has currently been described as bioactive plant compound. The objective of the study was to determine the role of associated phytic acid for the described lupin protein effects. A two-factorial study with ApoE knockout mice was conducted in which mice received lupin protein isolate or casein with or without phytase. Phytic acid was added to the casein diets to a final concentration identical to the lupin protein diets. Here we show that the serum concentrations of cholesterol, lathosterol and desmosterol were lower and the faecal bile acid excretion was higher in the groups fed lupin proteins than in the groups fed casein (p < 0.05). Mice that received the lupin protein diet containing phytic acid were characterized by a lower aortic calcification than mice of the other three groups (p < 0.05). In conclusion, our results show that the cholesterol lowering properties of lupin protein isolate were not caused by phytic acid. However, the hypocalcific action of lupin proteins appears to depend on the combination of lupin proteins and phytic acid.

Lupin protein isolate versus casein modifies cholesterol excretion and mRNA expression of intestinal sterol transporters in a pig model.

BACKGROUND: Lupin proteins exert hypocholesterolemic effects in man and animals, although the underlying mechanism remains uncertain. Herein we investigated whether lupin proteins compared to casein modulate sterol excretion and mRNA expression of intestinal sterol transporters by use of pigs as an animal model with similar lipid metabolism as humans, and cellular cholesterol-uptake by Caco-2 cells. METHODS: Two groups of pigs were fed cholesterol-containing diets with either 230 g/kg of lupin protein isolate from L. angustifolius or 230 g/kg casein, for 4 weeks. Faeces were collected quantitatively over a 5 d period for analysis of neutral sterols and bile acids by gas chromatographically methods. The mRNA abundances of intestinal lipid transporters were analysed by real-time RT-PCR. Cholesterol-uptake studies were performed with Caco-2 cells that were incubated with lupin conglutin γ, phytate, ezetimibe or albumin in the presence of labelled [4-14C]-cholesterol. RESULTS: Pigs fed the lupin protein isolate revealed lower cholesterol concentrations in total plasma, LDL and HDL than pigs fed casein (P < 0.05). Analysis of faeces revealed a higher output of cholesterol in pigs that were fed lupin protein isolate compared to pigs that received casein (+57.1%; P < 0.05). Relative mRNA concentrations of intestinal sterol transporters involved in cholesterol absorption (Niemann-Pick C1-like 1, scavenger receptor class B, type 1) were lower in pigs fed lupin protein isolate than in those who received casein (P < 0.05). In vitro data showed that phytate was capable of reducing the uptake of labelled [4-14C]-cholesterol into the Caco-2 cells to the same extend as ezetimibe when compared to control (-20.5% vs. -21.1%; P < 0.05). CONCLUSIONS: Data reveal that the cholesterol-lowering effect of lupin protein isolate is attributable to an increased faecal output of cholesterol and a reduced intestinal uptake of cholesterol. The findings indicate phytate as a possible biofunctional ingredient of lupin protein isolate.

Expression of genes involved in hepatic carnitine synthesis and uptake in dairy cows in the transition period and at different stages of lactation.

BACKGROUND: In rodents and pigs, it has shown that carnitine synthesis and uptake of carnitine into cells are regulated by peroxisome proliferator-activated receptor α (PPARA), a transcription factor which is physiologically activated during fasting or energy deprivation. Dairy cows are typically in a negative energy balance during early lactation. We investigated the hypothesis that genes of carnitine synthesis and uptake in dairy cows are enhanced during early lactation. RESULTS: mRNA abundances of PPARA and some of its classical target genes and genes involved in carnitine biosynthesis [trimethyllysine dioxygenase (TMLHE), 4-N-trimethylaminobutyraldehyde dehydrogenase (ALDH9A1), γ-butyrobetaine dioxygenase (BBOX1)] and uptake of carnitine [novel organic cation transporter 2 (SLC22A5)] as well as carnitine concentrations in liver biopsy samples of 20 dairy cows in late pregnancy (3 wk prepartum) and early lactation (1 wk, 5 wk, 14 wk postpartum) were determined. From 3 wk prepartum to 1 wk postpartum, mRNA abundances of PPARΑ and several PPARΑ target genes involved in fatty acid uptake, fatty acid oxidation and ketogenesis in the liver were strongly increased. Simultaneously, mRNA abundances of enzymes of carnitine synthesis (TMLHE: 10-fold; ALDH9A1: 6-fold; BBOX1: 1.8-fold) and carnitine uptake (SLC22A5: 13-fold) and the concentration of carnitine in the liver were increased from 3 wk prepartum to 1 wk postpartum (P < 0.05). From 1 wk to 5 and 14 wk postpartum, mRNA abundances of these genes and hepatic carnitine concentrations were declining (P < 0.05). There were moreover positive correlations between plasma concentrations of non-esterified fatty acids (NEFA) and hepatic carnitine concentrations at 1 wk, 5 wk and 14 wk postpartum (P < 0.05). CONCLUSIONS: The results of this study show for the first time that the expression of hepatic genes of carnitine synthesis and cellular uptake of carnitine is enhanced in dairy cows during early lactation. These changes might provide an explanation for increased hepatic carnitine concentrations observed in 1 wk postpartum and might be regarded as a physiologic means to provide liver cells with sufficient carnitine required for transport of excessive amounts of NEFA during a negative energy balance.

Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins.

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.

Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form.

Maltosine, a 3-hydroxy-4-pyridinone derivative of lysine formed in the course of the advanced Maillard reaction, is an effective metal chelating agent. It therefore represents an interesting compound for the treatment of metal ion storage diseases. We synthesized 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine (free maltosine) and its dipeptide derivatives alanylmaltosine (Ala-Mal) and maltosinylalanine (Mal-Ala) and examined the transepithelial flux of these compounds across Caco-2 cells and their interaction with membrane transporters. Transepithelial flux of maltosine was significantly higher when added as Ala-Mal and Mal-Ala than in free form. Assays at Caco-2 cells and at HeLa cells expressing the human peptide transporter (hPEPT)1 revealed that Ala-Mal and Mal-Ala show medium to high affinity to the system. Only free but not peptide-bound maltosine inhibited the uptake of l-[(3)H]lysine in Caco-2 and OK cells. Maltosine dipeptides were transported by hPEPT1 across cell membranes and accumulated in hPEPT1-transfected HeLa cells. In electrophysiological measurements at hPEPT1-expressing Xenopus laevis oocytes, Ala-Mal and Mal-Ala induced significant inward directed currents. We conclude that Ala-Mal and Mal-Ala are transported by hPEPT1 into intestinal cells and then hydrolyzed to free maltosine and alanine. The results suggest that the oral bioavailability of maltosine can be increased significantly by applying this drug candidate in peptide-bound form.

Transport of the advanced glycation end products alanylpyrraline and pyrralylalanine by the human proton-coupled peptide transporter hPEPT1.

The glycation compound pyrraline, which originates from the advanced Maillard reaction, appears in urine after consumption of pyrraline-containing food. We hypothesized that the absorption of pyrraline occurs in the form of dipeptides rather than the free amino acid. The human intestinal peptide transporter hPEPT1 was transiently expressed in HeLa cells. In hPEPT1-transfected cells but not in cells transfected with empty vector, the uptake of [(14)C]glycylsarcosine was strongly inhibited by alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala). Free pyrraline did not inhibit peptide uptake. In Xenopus laevis oocytes expressing human PEPT1, both Ala-Pyrr and Pyrr-Ala generated significant inward directed currents. In a third approach, uptake of the dipeptides into hPEPT1-transfected HeLa cells was analyzed by HPLC. Ala-Pyrr and Pyrr-Ala were taken up by hPEPT1-expressing cells at a 4- to 7-fold higher rate than by HeLa cells transfected with the empty vector. We conclude that pyrraline containing dipeptides are transported by hPEPT1 in an electrogenic manner into intestinal cells.

The bioactive dipeptide anserine is transported by human proton-coupled peptide transporters.

The bioactive dipeptide derivative anserine (beta-alanyl-1-N-methyl-L-histidine) is absorbed from the human diet in intact form at the intestinal epithelium. The purpose of this study was to investigate whether anserine is a substrate of the H(+)/peptide cotransporters 1 and 2 (PEPT1 and PEPT2). We first assessed the effects of anserine on [(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hypertensive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2. Anserine inhibited [(14)C]Gly-Sar uptake with K(i) values of 1.55 mM (Caco-2) and 0.033 mM (SKPT). In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, K(i) values of 0.65 mM (hPEPT1) and 0.18 mM (hPEPT2) were obtained. We conclude from these data that anserine is recognized by PEPT1 and PEPT2. Carnosine also inhibited [(14)C]Gly-Sar uptake. Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine. We conclude that anserine and carnosine interact with the human intestinal peptide transporter and are transported by hPEPT1 in an active, electrogenic H(+) symport. As PEPT1 is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. In addition, anserine might be useful for the design of new substrates of peptide transporters, such as prodrugs, that can be administered orally.

Transport of free and peptide-bound pyrraline at intestinal and renal epithelial cells.

Pyrraline is a quantitatively dominating glycation compound of the advanced Maillard reaction in foods and can be found in urine after consumption of pyrraline-containing food items. The purpose of this study was to investigate the transport of pyrraline and its dipeptide derivatives alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala) at intestinal and renal cell lines. Pyrraline inhibited the l-[(3)H]lysine uptake with IC(50) values of 0.3 mM (Caco-2 cells) and 3.5 mM (OK cells), respectively, but not the uptake of [(14)C]Gly-Sar (Caco-2 and SKPT cells). In contrast, Ala-Pyrr strongly inhibited the uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells with IC(50) values of 0.19 and 0.017 mM, respectively. Pyrr-Ala inhibited the carrier-mediated uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells by 50% at concentrations of 0.03 and 0.008 mM, respectively. The transepithelial flux of peptide-bound pyrraline across Caco-2 cell monolayers was up to 15-fold higher compared to the flux of free pyrraline. We conclude that free pyrraline is not a substrate for the intestinal lysine transporter and that the absorption of dietary pyrraline occurs most likely in the form of dipeptides rather than as the free amino acid.

Clofibrate treatment up-regulates novel organic cation transporter (OCTN)-2 in tissues of pigs as a model of non-proliferating species.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P<0.05). Pigs treated with clofibrate had also higher concentrations of free and total carnitine in the liver and a higher concentration of free carnitine in skeletal muscle than control pigs (P<0.05). Concentrations of gamma-butyrobetaine, the precursor of endogenous formation of carnitine, in liver, muscle and plasma did not differ between both groups; the activity of gamma-butyrobetaine dioxygenase, the rate limiting enzyme of carnitine synthesis, in the liver was lower in pigs treated with clofibrate than in control pigs (P<0.05). This study shows for the first time that treatment with a PPAR alpha agonist causes an up-regulation of OCTN2 in liver, muscle and enterocytes from small intestine of pigs. This in turn increases carnitine concentrations in liver and muscle probably by enhancing carnitine uptake into cells.