Neville H Mcclenaghan

Siobhan M Scullion

Brian Mion

Chandralal Hewage

Paul G Malthouse

Peter R Flatt

Philip Newsholme

Dr Lorraine Brennan
email: lorraine.brennan@ucd.ie.

Prolonged L-alanine exposure induces changes in metabolism, Ca2+ handling and desensitization of insulin secretion in clonal pancreatic beta-cells

Keywords: alanine, insulin secretion, pancreatic beta cells, desensitization nuclear magnetic resonance, NMR, c-Jun N-terminal kinase, JNK, glyceraldehyde-3phosphate dehydrogenase, GAPDH

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Introduction

Insulin secretion from pancreatic beta-cells is tightly regulated by circulating glucose, and modulated by a number of physiological and pharmacological factors [START_REF] Robertson | Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes[END_REF][START_REF] Mcclenaghan | Physiological and pharmacological regulation of insulin release: insights offered through exploitation of insulin-secreting cell lines[END_REF][START_REF] Deeney | Metabolic control of beta-cell function[END_REF][START_REF] Mcclenaghan | Sharpey-Schafer Prize Lecture. Physiological regulation of the pancreatic beta-cell: Functional insights for understanding and therapy of diabetes[END_REF]. Amino acids represent an important class of modulators of pancreatic beta-cell function, and specific individual or mixtures of amino acids regulate insulin secretion both in vitro and in vivo. Pancreatic beta-cells are equipped with a range of specific amino acid transporters many of which are Na + dependent [5,[START_REF] Prentki | Neutral amino acid transport in isolated rat pancreatic islets[END_REF]. Three principal modes of insulinotropic action of amino acids have been characterized [START_REF] Yada | Action mechanisms of amino acids in pancreatic beta cells[END_REF]8]. Firstly, direct membrane depolarization resulting from beta-cell transport of cationic amino acids (such as arginine) promotes Ca 2+ -channel opening and insulin release. Other metabolizable amino acids (such as leucine) can increase intracellular ATP evoking closure of ATP-sensitive K + channels (K ATP channels), membrane depolarization and Ca 2+ influx stimulating insulin release. In addition to these mechanisms, certain amino acids including alanine may stimulate insulin release through both metabolism and as a direct result of the membrane depolarizing actions of Na + co-transport triggering Ca 2+ influx and ultimately insulin release. While the insulinotropic effects of arginine and leucine have long been known [START_REF] Sener | Stimulus-secretion coupling of arginine-induced insulin release: comparison between the cationic amino acid and its methyl ester[END_REF][10][START_REF] Herchuelz | Mechanism of argininestimulated Ca2+ influx into pancreatic B cell[END_REF], the importance of alanine in acute regulation of beta-cell metabolism and function has only been appreciated more recently [12][START_REF] Dixon | A comparative study of amino acid consumption by rat islet cells and the clonal beta-cell line BRIN-BD11 -the functional significance of L-alanine[END_REF][START_REF] Sener | The stimulus-secretion coupling of amino acid-induced insulin release. Insulinotropic action of L-alanine[END_REF]. Of particular note, is the positive effect of the abundant amino acid alanine on glucose metabolism and insulin secretion [12] suggesting that this amino acid may have a significant impact on beta-cells.

While acute exposure to nutrients and other insulinotropic agents exert positive beta-cell actions, prolonged exposure may induce desensitization and other detrimental effects [START_REF] Robertson | Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes[END_REF][START_REF] Deeney | Metabolic control of beta-cell function[END_REF][START_REF] Mcclenaghan | Sharpey-Schafer Prize Lecture. Physiological regulation of the pancreatic beta-cell: Functional insights for understanding and therapy of diabetes[END_REF][START_REF] Ball | Desensitization of sulphonylurea-and nutrient-induced insulin secretion following prolonged treatment with glibenclamide[END_REF]. Glucose is the principal physiological regulator of insulin release but chronic exposure to high levels of this sugar is associated with beta-cell deterioration, so-called glucose desensitization and glucotoxicity perhaps involving oxidative stress [START_REF] Bindokas | Visualizing superoxide production in normal and diabetic rat islets of Langerhans[END_REF][START_REF] Tanaka | A role for glutathione peroxidase in protecting pancreatic beta cells against oxidative stress in a model of glucose toxicity[END_REF][18], but this has been questioned [START_REF] Martens | Glucose suppresses superoxide generation in metabolically responsive pancreatic beta cells[END_REF][START_REF] Khaldi | Antioxidants Nacetyl-L-cysteine and manganese(III)tetrakis (4-benzoic acid)porphyrin do not prevent beta-cell dysfunction in rat islets cultured in high glucose for 1 wk[END_REF]. Similarly, free fatty acids act as important signalling molecules and beta-cell fuels, enhancing insulin release, but long-term exposure can induce beta-cell lipotoxicity particularly in the presence of high glucose levels [21]. A recent metabolomic analysis of urine from type 2 diabetic subjects and animals models showed that the main metabolic disturbances occurred in amino acid metabolism with an increased excretion of glutamine, glutamate, alanine, taurine and ornithine for human subjects [START_REF] Salek | A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human[END_REF]. The long-term consequences of such altered amino acid concentrations on the pancreatic beta cell has received little attention to date.

A recent study indicated that prolonged exposure to the amino acid, alanine, induced upregulation of gene expression of certain metabolic and signal transduction elements coupled with enhanced protection against pro-inflammatory cytokine-induced apoptosis [23]. Notably, these preliminary studies indicated an alteration in beta-cell responsiveness subsequent amino acid-induced stimulation [23]. This observation prompted the present study examining in more detail the nature of the demise in insulin secretion and beta-cell function following prolonged alanine exposure, demonstrating induction of reversible alterations to metabolic flux, calcium handling and insulin secretion.

Materials and methods

Reagents [3- 13 C]alanine was obtained from Goss Scientific (Great Baddow, Essex, UK.). 125 I-bovine insulin was purchased from Amersham Biosciences (Bucks, UK). All other chemicals were obtained from Sigma-Aldrich Chemical Company (Poole, UK) and BDH chemicals (Poole, UK). Culture media and foetal bovine serum were obtained from Gibco (Glasgow, UK). Membrane potential kit and calcium assay kit were purchased from Molecular Devices (Sunnyvale, CA, USA).

Cell culture and treatment with L-alanine for NMR studies

Experiments utilized glucose-and amino acid-responsive clonal pancreatic BRIN-BD11 beta-cells [START_REF] Mcclenaghan | Sharpey-Schafer Prize Lecture. Physiological regulation of the pancreatic beta-cell: Functional insights for understanding and therapy of diabetes[END_REF][START_REF] Mcclenaghan | Engineering cultured insulin-secreting pancreatic B-cell lines[END_REF]. These cells have proven to be particularly useful as model beta-cells for studies involving NMR based experiments which require substantial cellular mass [12,[START_REF] Brennan | 13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line[END_REF]. The origin of BRIN-BD11 cells are described elsewhere [START_REF] Mcclenaghan | Sharpey-Schafer Prize Lecture. Physiological regulation of the pancreatic beta-cell: Functional insights for understanding and therapy of diabetes[END_REF][START_REF] Mcclenaghan | Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion[END_REF]. These cells provide an appropriate beta-cell model as evidenced by studies of insulin secretion [START_REF] Mcclenaghan | Sharpey-Schafer Prize Lecture. Physiological regulation of the pancreatic beta-cell: Functional insights for understanding and therapy of diabetes[END_REF][START_REF] Mcclenaghan | Engineering cultured insulin-secreting pancreatic B-cell lines[END_REF], electrophysiology, Ca 2+ handling [START_REF] Van Eylen | Overexpression of the Na/Ca exchanger shapes stimulus-induced cytosolic Ca(2+) oscillations in insulin-producing BRIN-BD11 cells[END_REF] and cellular defense [START_REF] Mcclenaghan | Sharpey-Schafer Prize Lecture. Physiological regulation of the pancreatic beta-cell: Functional insights for understanding and therapy of diabetes[END_REF]. In addition, signalling, insulin secretory and cell viability responses to glucose, amino acids as well as other stimuli have been well characterized [START_REF] Mcclenaghan | Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion[END_REF][START_REF] Van Eylen | Overexpression of the Na/Ca exchanger shapes stimulus-induced cytosolic Ca(2+) oscillations in insulin-producing BRIN-BD11 cells[END_REF][START_REF] Chapman | ATP-sensitive potassium channels and efaroxan-induced insulin release in the electrofusion-derived BRIN-BD11 beta-cell line[END_REF][START_REF] Kamagate | Plasma membrane Ca(2+)-ATPase overexpression reduces Ca(2+) oscillations and increases insulin release induced by glucose in insulinsecreting BRIN-BD11 cells[END_REF]. BRIN-BD11 cells were grown and maintained in RPMI-1640 tissue culture medium with 10% (v/v) foetal bovine serum, 0.1% antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin) and 11.1 mmol/l D-glucose in a 37 ºC in a humidified atmosphere of 5% CO 2 and 95% air as described previously [START_REF] Mcclenaghan | Characterization of a novel glucose-responsive insulin-secreting cell line, BRIN-BD11, produced by electrofusion[END_REF].

For experiments investigating prolonged exposure to L-alanine, cell monolayers were maintained in T175cm 2 flasks (Greiner, Germany) and treated for 18 h in the presence of 10 mmol/l Lalanine. A concentration of 10 mmol/l was used as this provides a robust and reproducible stimulus for insulin secretion experiments [30]. For comparison cells were grown in parallel in the absence of Lalanine (Standard culture; Control). Cells were then washed with phosphate buffered saline and preincubated at 37 ºC for 20 min in Krebs-Ringer Bicarbonate (KRB) buffer comprising 115 mmol/l NaCl, 4.7 mmol/l KCl, 1.28 mmol/l CaCl 2 , 1.2 mmol/l KH 2 PO 4 , 1.2 mmol/l MgSO 4 .7H 2 O, 10 mmol/l NaHCO 3 , 5 g/l bovine serum albumin (pH 7.4) supplemented with 1.1 mmol/l D-glucose. This was followed by 1 h incubation with KRB test buffer containing 1.1 mmol/l glucose and 10 mmol/l [3-13 C]alanine and aliquots of test buffer was removed and stored at -20 ºC for further analysis. After test exposures metabolites were extracted from cells using a perchloric acid extraction procedure. Briefly, cells were washed with ice-cold phosphate-buffered saline, perchloric acid (6%) added and cells scraped from tissue culture flasks. Extracts of six culture flasks (approximately 10 8 cells) were pooled, centrifuged at 200 g and resulting supernatants neutralised with KOH (5 M and 0.1 M solutions) before soaking pellets overnight in 0.1 M NaOH. The protein concentration was determined using the Lowry method [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] and neutralised supernatant was centrifuged again (at 200 g), and supernatant treated with Chelex-100 resin before lyophilization. Each experiment was carried out on at least three independent cultures of the BRIN-BD11 cells. Lyophilised cell extracts were dissolved in 3 ml potassium phosphate buffer (100 mmol/l; pH 7.0), centrifuged and supernatant carefully removed before addition of 10% D 2 O and pH checked and adjusted where necessary with 0.1 M NaOH or 0.1 M HCl. NMR analyses were then performed as detailed below.

NMR analyses

For 13 C NMR experiments, an insert containing 5 % v/v dioxane in water was used as an external signal intensity reference. A solution of L-alanine, L-glutamate, lactate and D-glucose, each at a concentration of 100 mmol/l, was prepared and used to quantitate concentrations of metabolites in the 13 C spectra. Proton decoupled 13 C spectra were acquired on a Bruker DRX 500 spectrometer using a 10 mm broadband probe. Typically spectra were acquired with 32 K data points using 90º pulses, 260 ppm spectral width, 2.5 s relaxation delay and 12000 -20000 scans. Spectra were recorded at 25 ºC. A c c e p t e d M a n u s c r i p t Chemical shifts were referenced to tetramethylsilane at 0 ppm. Data were processed with no zero filling using Bruker WINNMR software and exponential multiplications with 2 Hz line broadening were performed. Assignments of intermediate metabolites were made by comparison with chemical shift tables in the literature or by addition of 100 mmol/l of unlabelled amino acid. The amount of 13 C in each resonance was evaluated by integration of the extract peaks and the corresponding peaks in the standard sample relative to the dioxane signal. Corrections for the natural abundance signal were made. In the case of the aspartate peaks the amount of 13 C was estimated by use of the integrals and the known dioxane concentration. The contribution of the individual isotopomers was assessed using the deconvolution routines in WINNMR. The absolute enrichments of the L-glutamate were related to the glutamate concentration in the extracts to give the specific enrichments.

Determination of insulin release and cellular content

Insulin release was determined from BRIN-BD11 cells monolayers. Briefly, cells were harvested, resuspended in culture medium and seeded in each well of 24-well multiplates at a density of 1.5 x 10 5 cells per well. After overnight incubation and attachment at 37 ºC, standard RPMI-1640 tissue culture medium was replaced with standard culture media (no L-alanine) or culture media supplemented with 10 mmol/l L-alanine and cells returned to culture. After 18 h, culture media was removed from each well and replaced with 1 ml KRB buffer supplemented with 0.1 % (w/v) bovine serum albumin and 1.1 mmol/l D-glucose, for a 20 min pre-incubation at 37 ºC. At the end of the pre-incubation period, KRB pre-incubation buffer was removed and replaced with 1 ml of KRB test buffer supplemented with 1.1 mmol/l glucose and 10 mmol/l L-alanine as indicated in the Figures. After 60 min incubation, aliquots of test buffer were removed from each well and stored at -20 o C. Cellular insulin was extracted by addition of 1 ml of ice-cold acid-ethanol solution (75% v/v ethanol, 1.5% v/v concentrated HCl). Insulin was measured from each sample by dextran-charcoal radioimmunoassay [START_REF] Flatt | Abnormal plasma glucose and insulin responses in heterozygous lean (ob/+) mice[END_REF], using guinea-pig anti-porcine insulin serum, and rat insulin standard.

Assessment of cellular integrity following alanine culture

Following culture in the absence or presence of alanine, cellular integrity was assessed by means of either the neutral red or MTT (dimethyl thiazolyl blue tetrazolium bromide) assay. For the neutral red assay 2 x 10 4 cells were seeded into each well of 96 well plates and cultured for 18 h in the absence or presence of 10 mmol/l alanine. Cells were then exposed to 100 µl of neutral red solution (PBS, 0.05 mg/ml neutral red) for 2 h, and after washing with PBS, 100 µl of glacial acetic acid solution (50 ml ethanol, 1 ml glacial acetic acid, 49 ml dH 2 O) was added to each well, plates were shaken for 15 min for cell lysis and neutral red release from cells. Absorbance from each well was then recorded at 540 nm and compared to control (culture in absence of 10 mmol/l alanine) and viability expressed as a percentage relative to control. For MTT analyses, 2 x 10 4 cells were seeded into each well of 96 well plates and cultured for 18 h in the absence or presence of 10 mmol/l alanine before incubation with 100 µl MTT solution (1 : 10 dilution of the 5 mg/ml stock -MTT in RPMI without phenol red) at 37 °C for 30 min. MTT solution was then removed and cells lysed with 100 µl DMSO (dimethylsulphoxide). Optical densities were recorded at 562 nm with a reference wavelength of 650 nm and cell viability expressed as a percentage relative to control.

ATP and protein expression determinations

For ATP and protein measurements the cells were seeded in 6 well plates (1 x 10 6 cells) and allowed to form monolayers overnight. Cells were then exposed to culture in the absence or presence of 10 mmol/l L-alanine for 18 h followed by a 1 h incubation in KRB buffer, as described earlier. Following incubation cells were lysed with a somatic cell ATP-releasing reagent and placed on ice [START_REF] Nguyen | Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells[END_REF]. Intracellular ATP was measured using a luciferin/luciferase-based assay (Biaffin GmbH) ice [START_REF] Nguyen | Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells[END_REF]. Protein expression was examined using Western blotting where cells were washed with ice cold PBS before lysis in ripa buffer (0.5 M Tris-HCl pH7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 nM EDTA). Samples were centrifuged at 12,000 g for 15 min at 4 °C, supernatant collected and total protein determined using a BCA assay. Samples were then subjected to SDS-PAGE electrophoresis using a 12.5 % resolving gel after which resolved proteins were transferred to nitrocellulose membranes and blocked for 1 h at room temperature with Tris-buffered saline supplemented with 0.1 % Tween and 5 % non-fat powdered milk. Nitrocellulose blots were incubated for 2 h with the pyruvate dehdyrogenase kinase-2 (PDK-2) polyclonal antibody (Abgent) or 4 h with the PDK-4 polyclonal antibody, washed and incubated with horseradish peroxidase (Santa Cruz Biotechnology). Bound antibody was visualised by using ECL according to the manufacturers instructions (Pierce, Rockford, IL, USA). Equal loading was varied by analysis of total c-Jun Nterminal kinase (JNK) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blots were then exposed to Hyperfilm and bands quantified by scanning densitometry.

Semi-quantitative analyses of PDK-2 and PDK-4 gene expression

For semi-quantitative detection of PDK-2 and PDK-4 mRNA levels, cells were grown and maintained in 6-well plates. Total RNA was extracted using the thiocyanate-phenol-chloroform method using Tri reagent according to the manufacturers protocol (Molecular Research Centre Inc., Cincinnati). Complementary DNA (cDNA) was synthesized and amplified by PCR using primers for PDK-2, PDK-4 and 18S (housekeeping gene). The following primer sequences were used: PDK-2 forward-AGTTCACTGATGCCCTGGTC, PDK-2 reverse-GTTGGTGGCATTGACTTCCT, PDK-4 forward-CGTCGCCAGAATTAAAGCTC, PDK-4 reverse-TAACCAAAACCAGCCAAAGG. PCR was carried out according to the following protocol: 2 min denaturing step at 94 °C followed by 25-30 cycles of amplification (45 s denaturation at 94 °C, 45 s annealing at 60 °C, 45 s extension at 72 °C) and a final extension step of 2 min at 72 °C. PCR products were analysed on ethidium bromide-stained agarose gels, photographed under UV transillumination and data expressed relative to housekeeping gene 18S.

Determination of metabolite levels

Alanine concentrations were determined in the media following the incubation periods using an enzymatic-based reaction coupled to the production of NADH. Total glutamate and lactate levels were determined using a YSI 7100 amino acid analyzer.

Measurement of membrane potential and intracellular calcium

Membrane potential and intracellular calcium ([Ca 2+ ] i ) were determined using monolayers of BRIN-BD11 cells [START_REF] Miguel | Timecorrelation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR[END_REF]. Cells were seeded into 96-well black-walled, clear bottom microplates at a density of 1.0 x 10 5 cells per well and allowed to attach overnight, before replacing culture media with either standard culture media or 10 mmol/l alanine-supplemented culture media. After 18 h culture, media in each well was replaced with 100 µl of KRBB and monolayers incubated for 20 min after which 100 µl of either FLEX membrane potential assay kit [35] or FLEX calcium assay kit (Molecular Devices, CA, USA), was added to wells at 37 ºC as previously described [START_REF] Miguel | Timecorrelation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR[END_REF]. Fluorometric data were acquired using the FLEXstation TM scanning fluorometer and integrated fluid transfer workstation (Molecular Devices, CA, USA). The cells were exposed to excitation light from a xenon-arc flashlamp at a wavelength of 530 nm (membrane) or 485 nm (calcium) and subsequent fluorescence emission measured at 565 nm (membrane) or 525 nm (calcium) using a bottom read mode. Emission cut-off filters were set at 550 nm for membrane potential or 515 nm for [Ca 2+ ] i .

Results

Effects of prolonged exposure to alanine on cellular integrity, insulin secretory responses and cellular insulin content

Analysis of cellular integrity, by neutral red and MTT assays, following culture of cells in the absence or presence of 10 mmol/l alanine revealed that alanine culture did not significantly alter cellular integrity compared with standard culture (Figure 1). The insulin-secretory responses to 10 mmol/l alanine after 18 h culture in the absence or presence of 10 mmol/l alanine are given in Figure 2A. As illustrated, while prior exposure to 10 mmol/l alanine did not significantly alter basal insulin release at 1.1 mmol/l glucose, alanine culture markedly suppressed the subsequent stimulatory effect of acute addition of alanine compared with control cells (74% reduction, p<0.001). This alanine-induced demise in insulin output was partially restored (p<0.01) after a further 18 h recovery period involving culture in absence of alanine. As shown in Figure 2B, 18 h exposure to 10 mmol/l alanine in culture did not significantly affect insulin content compared with control cells exposed to standard culture medium, and there was evidence of enhanced content after an 18 h recovery period. Taken together, these data indicate alanine-induced beta-cell desensitization.

It was also of interest to assess if the observed desensitization was specific to beta-cell alanine responsiveness, by assessing the impact of prolonged exposure to alanine on subsequent responses to a depolarizing concentration of KCl or other key nutrient secretagogues (Figure 3). Alanine culture did not alter the marked 6.7-fold (p<0.001) acute insulinotropic action of 30 mmol/l KCl (Figure 3A). Similarly, the insulin-releasing effects of acute stimulatory 16.7 mmol/l glucose, 10 mmol/l mannose or 30 mmol/l KIC were not affected by alanine culture (Figure 3B). To probe the alanine-induced desensitization mechanism further cells were cultured with the non-metabolizable amino acid analogue aminoisobutyric acid (AIB) which shares the same transport mechanism as alanine (Figure 4). As with alanine culture, prolonged exposure to AIB in culture did not alter basal insulin release at 1.1 mmol/l glucose. Interestingly, alanine culture also served to significantly reduce subsequent responsiveness to AIB (by 44%, p<0.05), although not to the same extent as the reduction in alanine-induced insulin output (Figure 4). Likewise, AIB culture served to decrease alanine-induced insulin release (by 71%, p<0.001) and the insulinotropic response to AIB (by 63%, p<0.01) (Figure 4). While alanine or AIB culture also reduced serine-induced insulin secretion compared with standard culture (p<0.01) (Figure 4).

Effects of prolonged exposure to alanine on membrane potential and intracellular calcium

The impact of culture with 10 mmol/l alanine on subsequent membrane potential and [Ca 2+ ] i responses to acute stimulation with alanine (10 mmol/l) are illustrated in Figure 5. As shown, after standard culture in the absence of alanine, BRIN-BD11 cells demonstrated a marked membrane depolarization, but area-under-the-curve (AUC) analysis revealed that this increase in membrane potential was significantly (p<0.01) suppressed in cells exposed to alanine culture (Figure 5A), although membrane potential at 1.1 mmol/l glucose was not altered (data not shown). The suppressed depolarization response to acute stimulation with 10 mmol/l alanine was partly reversed after an 18 h recovery period consistent with a partial restoration of insulin release.

Corresponding to the membrane depolarizing effect of alanine, after standard culture, acute stimulation with 10 mmol/l alanine caused a notable rise in [Ca 2+ ] i consistent with the insulin-releasing actions of this amino acid (Figure 5B). However, AUC analysis also demonstrated a significant (p<0.01) reduction in the 10 mmol/l alanine-induced rise in [Ca 2+ ] i following alanine culture (Figure 5B). At 1.1 mmol/l glucose [Ca 2+ ] i levels were similar following culture in presence or absence of alanine (data not shown). Again, the reduced rise in [Ca 2+ ] i following alanine culture was partially restored by 18 h recovery in culture medium in the absence of alanine, consistent with the view that the alanine-induced beta-cell suppression was a reversible desensitization.

Effects of prolonged exposure to alanine on subsequent alanine metabolism

Following incubation with [3-13 C]alanine, the end products included glutamate labelled at positions C2, C3 and C4 and aspartate labelled at position C2 and C3 (Figure 6). The glutamate C4 peak consisted predominantly of a singlet peak due to enrichment at the C4 position only (Figure 6A). A small doublet peak was also present, which was due to glutamate labelled at position C3 and C4 (Figure 6A). Following 18 h alanine culture there was a significant increase in glutamate labelled at position C4 but not at position C2 and C3 (Figure 6A and Figure 6B). Closer examination of the C4 peak revealed that the C4 singlet (C4S) increased significantly from 18.2 ± 1.9 to 29.6 ± 1.7 nmol/mg protein (P<0.005). The singlet at C4 (C4S) can only be labelled by entry via pyruvate dehydrogenase (PDH), indicating increased flux through PDH following prolonged alanine exposure [36].

Calculation of the specific enrichment showed that there was no change for glutamate position C2 and C3 (Figure 6C). However, the percentage enrichment increased in position C4 significantly (Figure 6C). The amount of 13 C labelled alanine in the extracts did not significantly change following prolonged exposure to alanine (0.53 ± 0.01 µmol/mg protein for control conditions and 0.49 ± 0.04 µmol/mg protein for alanine culture). Interestingly, lactate release did not change significantly from control conditions during the 1 h incubation (25.7 ± 1.8 nmol/mg protein for control conditions, and 22.5 ± 3.6 nmol/mg protein for alanine culture). Furthermore, the amount of L-alanine remaining in the media at the end of the 1 h incubation was not significantly different following prolonged culture with L-alanine (17.1 ± 3.1 µmol/ mg protein for control conditions, and 11.7 ± 4.0 µmol/mg protein for alanine culture).

Intracellular ATP levels were measured in parallel experiments at the end of the 1 h incubation and under control conditions the ATP concentration was 5.8 pmol/10 4 cells. Alanine culture significantly (p<0.05) reduced the ATP concentration by 28 % to 4.2 pmol/10 4 cells.

Effects of prolonged exposure to alanine on PDK-2 and PDK-4 gene and protein expression

18 h exposure of BRIN-BD11 cells to alanine in culture resulted in a reduction in pyruvate dehydrogenase kinase-2 (PDK-2) protein expression as evaluated by Western blot analysis (Figure 7A,B). Consistent with this alteration in protein expression, semi-quantitative reverse transcription-PCR analysis of PDK-2 gene expression was also suppressed by alanine culture (Figure 7C). Similarly, as shown in Figure 8A, alanine culture resulted in reduced pyruvate dehydrogenase kinase-4 (PDK-4) protein expression and mRNA levels of PDK-4 (Figure 8B). These observations are consistent with the altered flux through PDH observed by the NMR experiments.

Discussion

Prolonged exposure of beta cells to glucose, fatty acids and insulinotropic drugs has been shown to induce beta-cell desensitization to subsequent acute stimulation [START_REF] Robertson | Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes[END_REF][START_REF] Ball | Desensitization of sulphonylurea-and nutrient-induced insulin secretion following prolonged treatment with glibenclamide[END_REF]21]. Most recently, the effects of prolonged (24 h) exposure to alanine or glutamine on gene expression has been reported, revealing an alteration of 66 or 166 genes, respectively, with a large functional core related to signalling and metabolism [23,[START_REF] Corless | Glutamine regulates expression of key transcription factor, signal transduction, metabolic gene, and protein expression in a clonal pancreatic beta-cell line[END_REF]. These studies also indicated that prolonged alanine exposure may confer protection against cytokine-induced beta-cell apoptosis. However, while the detrimental actions of glucose and lipids on beta-cell function are well characterized the consequences of prolonged amino acid exposure on beta-cell metabolism, insulin secretion and function has been largely neglected, prompting the present studies. The importance of the present study is emphasised by the fact that alanine is quantitatively the second most abundant amino acid in blood and extracellular tissues in vivo.

Following 18 h culture with alanine there was a marked impairment to subsequent characteristic potent insulin-releasing actions of this amino acid. Alanine culture did not however significantly diminish basal insulin release or cellular insulin content. Furthermore, when cells exposed to alanine for 18 h were cultured for a further 18 h in the absence of this amino acid, there was a partial restoration of alanine-induced insulin release. This would indicate a reversible desensitization as previously reported with other insulinotropic agents [START_REF] Ball | Desensitization of sulphonylurea-and nutrient-induced insulin secretion following prolonged treatment with glibenclamide[END_REF][START_REF] Mcclenaghan | Induced desensitization of the insulinotropic effects of antidiabetic drugs, BTS 67 582 and tolbutamide[END_REF][START_REF] Mcclenaghan | Specific desensitization of sulfonylureabut not imidazoline-induced insulin release after prolonged tolbutamide exposure[END_REF], Consistent with a specific alanineinduced amino acid desensitization, responses to depolarizing concentration of KCl, stimulatory glucose, the insulinotropic hexose sugar mannose, or the 2-keto acid KIC were not affected by chronic alanine culture.

The mode of action by which alanine stimulates insulin release involves both co-transport into cells with Na + and rapid intracellular metabolism to generate ATP and other metabolic signalling factors [12-14, 40, 41]. The insulinotropic response to a non-metabolizable amino acid analogue of alanine, AIB was significantly reduced following alanine culture albeit to a lesser extent than alanineinduced insulin output. Similarly, AIB culture significantly impaired both alanine-and AIB-induced insulin release which would further indicate desensitization of the amino acid -Na+ co-transport system. However, it should be noted the acute uptake of alanine did not change following prolonged culture with alanine as indicated by the amount of 13 C labelled alanine in the extracts. In the present studies, alanine induced a rapid membrane depolarization, reaching a maximum peak around 30 sec and this response was markedly decreased in cells cultured for 18 h with alanine. The depolarizing action of alanine is consistent with acute effects of this and other amino acids co-transported with Na + [START_REF] Mcclenaghan | Na+ cotransport by metabolizable and nonmetabolizable amino acids stimulates a glucose-regulated insulin-secretory response[END_REF]. Such amino acids evoke membrane depolarization and increase Ca 2+ influx, while metabolizable amino acids (including L-alanine) will additionally increase intracellular ATP concentration and other signalling factors, prompting K ATP channel closure and insulin exocytosis [30]. Indeed, the initial rapid rise in membrane potential observed here indicates potent membrane depolarizing actions, largely attributable to Na + co-transport and accumulation, while the sustained plateau likely reflects the actions of cellular metabolic signalling factors including ATP acting through K ATP channels and other signal elements to modulate [Ca 2+ ] i and insulin release [START_REF] Mcclenaghan | Na+ cotransport by metabolizable and nonmetabolizable amino acids stimulates a glucose-regulated insulin-secretory response[END_REF].

Consistent with other studies, acute exposure to alanine resulted in a rapid rise in intracellular Ca 2+ concentration [START_REF] Miguel | Timecorrelation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR[END_REF][START_REF] Asada | Identification of alpha-and beta-cells in intact isolated islets of Langerhans by their characteristic cytoplasmic Ca2+ concentration dynamics and immunocytochemical staining[END_REF]. However, in cells cultured with alanine the maximal [Ca 2+ ] i peak was markedly reduced correlating with suppressed membrane depolarization, an effect which was partially reversed following an 18 h recovery period, again suggesting non-permanent alanine-induced desensitization with prolonged exposure. While the rise in [Ca 2+ ] i reflects co-transport with Na + and metabolic ATP generation, the decreased response following prolonged alanine exposure prompted additional studies investigating L-alanine metabolism and ATP generation in cells cultured with Lalanine.

To explore the relevance of metabolic changes to the diminished insulin-secretory responses to alanine following prolonged alanine exposure, metabolism was traced using 13 C NMR analyses. The authors have previously utilized this approach to characterize nutrient metabolism taking advantage of cultured pancreatic BRIN-BD11 beta cells to enhance understanding of complex metabolic pathways [12,[START_REF] Brennan | 13C NMR analysis reveals a link between L-glutamine metabolism, D-glucose metabolism and gamma-glutamyl cycle activity in a clonal pancreatic beta-cell line[END_REF][START_REF] Patterson | Detrimental actions of metabolic syndrome risk factor, homocysteine, on pancreatic beta-cell glucose metabolism and insulin secretion[END_REF][START_REF] Brennan | Investigation of the effects of sulfonylurea exposure on pancreatic beta cell metabolism[END_REF]. The present studies revealed a significant increase in flux through PDH following 18 h alanine culture, indicated by an increase in the C4S peak of glutamate. Interestingly, this increased flux through PDH should subsequently lead to an increased NADH concentration and subsequently enhanced ATP production. However, alanine culture resulted in a small but significant decrease in ATP concentration, despite a notable increase in oxidative metabolism. Nevertheless as ATP is an important modulator of the depolarizing and Ca 2+ -dependent insulin-releasing actions of alanine, this observation is consistent with data arising from other components of this study. It should be noted that the changes in ATP levels did not affect the viability of the cells. Activity of the beta-cell Na + /K + pump has been reported to consume 75-80 % of basal energy production in these cells [START_REF] Malaisse | The stimulus secretion coupling of glucose-induced insulin release. XXVII. Effect of glucose on K+ fluxes in isolated islets[END_REF], and thus is it possible that the decrease in cellular ATP may be related to enhanced Na + /K + pump activity following prolonged alanine exposure. A study by Elmi and coworkers investigated the relationship between islet ATP content and pump activity [START_REF] Elmi | Modulation of islet ATP content by inhibition or stimulation of the Na(+)/K(+) pump[END_REF], demonstrating that pump inhibition with ouabain did not adversely affect ATP levels during acute incubations. However, on longer incubation a very different pattern emerged with decreased islet ATP concentration corresponding to reduced glucose oxidation, opposing the expected outcome of inhibition of the ATP consuming Na + /K + pump. Moreover, diazoxide has been demonstrated to reduce the basal activity of the pump and elevate islet ATP content [START_REF] Elmi | Relationships between the Na(+)/K(+) pump and ATP and ADP content in mouse pancreatic islets: effects of meglitinide and glibenclamide[END_REF], but the opposite has also been reported [START_REF] Grimmsmann | Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria[END_REF], making it difficult to draw clear conclusions on the relationship between ATP concentration and the activity of the Na + /K + pump. Considering that alanine is present in both our control and pre-cultured conditions and that acute alanine uptake did not change significantly it is unlikely that the reduction in ATP at the end of the 1 hr incubation is due to increased activity of the Na + /K + pump. However, it is possible that the observed alanine-induced decrease of ATP despite increased oxidative metabolism could result from an uncoupling of ATP production in mitochondria resulting from reactive oxygen species generated as a by-product of oxidative metabolism.

Given the observation that prolonged alanine exposure resulted in an increased flux through PDH it was of interest to examine the expression of major inhibitory pyruvate dehydrogenase kinases (PDK-2 and PDK-4). It should be noted that regulation of PDH activity is complex and that the PDKs represent only part of the regulation of PDH activity [49]. PDKs control PDH activity through phosphorylation, causing reduced enzyme activity [START_REF] Sugden | Selective modification of pyruvate dehydrogenase kinase isoform expression in rat pancreatic islets elicited by starvation and[END_REF]. Notably, several reports have linked altered PDH activity with a decline in beta-cell function, and expression of PDK-1, PDK-2 and PDK-4 has been reported in pancreatic beta-cells [START_REF] Sugden | Selective modification of pyruvate dehydrogenase kinase isoform expression in rat pancreatic islets elicited by starvation and[END_REF]51]. Phosphorylation of PDH occurs at three specific sites and PDK-2 exhibiting the highest activity at site 1 while PDK-4 has the highest activity towards site 2 [49]. The present data demonstrating down-regulation of PDK-2 and PDK-4 mRNA levels following alanine-culture, using semi-quantitative reverse transcription PCR are entirely consistent with the observed increased flux through PDH. Prolonged alanine culture also down-regulated PDK-2 and PDK-4 protein expression, again being consistent with an increased flux through PDH. Interestingly, a similar downregulation of PDK-4 was reported following exposure to high glucose [51] and upregulation of PDK-4 has been reported to lower PDH activity during starvation [START_REF] Sugden | Selective modification of pyruvate dehydrogenase kinase isoform expression in rat pancreatic islets elicited by starvation and[END_REF], allowing entry of acetyl-CoA derived from fatty acids.

Collectively, the present data demonstrate alanine-induced downregulation of important PDK isoforms in beta-cells, resulting in the observed increase in PDH activity / flux, which has the knock-on effect of reducing TCA cycle entry via PC and reduced entry of acetyl-CoA from fatty acid oxidation. Consequently, as PC flux is important for fuel-driven insulin secretion [52-54], the decreased flux through PC may at least partly account for the reduction in insulin secretion following prolonged alanine exposure. Further studies including a timecourse of the response are required to explore these possibilities, to test whether desensitization observed here with alanine and related AIB extends to other functionally important amino acids such as leucine and glutamine, and to evaluate the role of reactive oxygen species and other metabolic intermediates in amino acid-induced beta-cell desensitization. Notably, however the present studies clearly demonstrate that prolonged exposure to alanine can induce beta-cell desenstitization, impacting on cellular metabolism and amino acid regulation of cellular metabolic and ionic flux. Effects of prolonged exposure to alanine on subsequent alanine-induced insulin release (A) or cellular insulin content (B). BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine. An additional group examined the effects of a further 18 h incubation (recovery) on cells exposed to alanine using standard culture medium. For insulin secretion experiments (A), cells were pre-incubated (20 min) in buffer containing 1.1 mmol/l glucose, and the insulinotropic actions of 10 mmol/l alanine at 1.1 mmol/l glucose were tested during a 60 min incubation period. For examination of insulin content (B), after culture acid-ethanol extractions were performed prior to insulin radioimmunoassay analysis. Values are mean ± SEM (n=6). **p<0.01, ***p<0.001 compared with effects in the absence of alanine (None). ∆ p<0.05, ∆∆∆ p<0.001 compared with effect after standard culture. Ψ p<0.01, ΨΨ p<0.01 comparing effect following 18h recovery with effect after alanine culture. BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine then exposed for 1 h to the test agents. Cells were preincubated (1 h) in buffer containing 1.1 mmol/l glucose and the insulinotropic actions of 30 mmol/l KCl, 16.7 mmol/l glucose, 10 mmol/l mannose or 30 mmol/l 2-ketoisocaproic acid (KIC) were tested during a 60 min incubation. Effects of prolonged exposure to either 10 mmol/l alanine or 10 mmol/l 2-aminoisobutyric (AIB) on subsequent insulin responses to amino acid test agents. BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine or 10 mmol/l AIB then exposed for 1 h to the test agents. Cells were preincubated (1 h) in buffer containing 1.1 mmol/l glucose and the insulinotropic actions of 10 mmol/l alanine, 10 mmol/l AIB or 10 mmol/l serine were tested during a 60 min incubation. Value are mean ± SEM (n=6). *p<0.05, ***p<0.001 compared with effects in the absence of addition (None). ∆ p<0.05, ∆∆ p<0.01, ∆∆∆ p<0.001 compared with effects after standard culture. Area-under-the-curves (AUCs) for standard culture or alanine culture were 6704000 ± 388324 and 5077334 ± 304649, respectively. (B) Effects of prolonged exposure to alanine on alanine-induced changes in intacellular Ca 2+ in BRIN-BD11 cells at 1.1 mmol/l glucose. Each data point represents mean ± SEM (n=6). Area-under-the-curves (AUCs) for standard culture or alanine culture were 573527 ± 28720 and 374636 ± 47437, respectively.

Clinical

  Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

  Clinical Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t Statistical analysis Results are expressed as means ± SEM. Statistical significance was evaluated by using one-way ANOVA. Clinical Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

Clinical 1 .

 1 Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t Clinical Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t activation of peroxisome proliferator-activated receptor-alpha: implications for glucose-stimulated insulin secretion. Diabetes. 50, 2729-2736 51 Xu, J., Han, J., Epstein, P. N. and Liu, Y. Q. (2006) Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets. Biochem Biophys Res Commun. 344, 827-833 52 Lu, D., Mulder, H., Zhao, P., Burgess, S. C., Jensen, M. V., Kamzolova, S., Newgard, C. B. and Sherry, A. D. (2002) 13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS). Proc Natl Acad Sci U S A. 99, 2708-2713 53 Boucher, A., Lu, D., Burgess, S. C., Telemaque-Potts, S., Jensen, M. V., Mulder, H., Wang, M. Y., Unger, R. H., Sherry, A. D. and Newgard, C. B. (2004) Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and reversal with a malate analogue. J Biol Chem. 279, 27263-27271 54 Fransson, U., Rosengren, A. H., Schuit, F. C., Renstrom, E. and Mulder, H. (2006) Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets. Diabetologia. 49, 1578-1586 Clinical Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t Legends to Figures Figure The effects on cellular integrity of 18 h culture of 10 mmol/l alanine compared to control culture were assessed by neutral red assay as shown in Fig A. and by MTT assay as shown in Fig B. BRIN-BD11 cells were cultured in the presence or absence of 10 mmol/l alanine for 18 h and cell viability was assessed using the MTT and Neutral red assay. Values are expressed as a percentage of the control.

Figure 2 .

 2 Figure 2.Effects of prolonged exposure to alanine on subsequent alanine-induced insulin release (A) or cellular insulin content (B). BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine. An additional group examined the effects of a further 18 h incubation (recovery) on cells exposed to alanine using standard culture medium. For insulin secretion experiments (A), cells were pre-incubated (20 min) in buffer containing 1.1 mmol/l glucose, and the insulinotropic actions of 10 mmol/l alanine at 1.1 mmol/l glucose were tested during a 60 min incubation period. For examination of insulin content (B), after culture acid-ethanol extractions were performed prior to insulin radioimmunoassay analysis. Values are mean ± SEM (n=6). **p<0.01, ***p<0.001 compared with effects in the absence of alanine (None). ∆ p<0.05, ∆∆∆ p<0.001 compared with effect after standard culture. Ψ p<0.01, ΨΨ p<0.01 comparing effect following 18h recovery with effect after alanine culture.

Figure 3 .

 3 Figure 3. Effects of prolonged exposure to alanine on insulin secretion in response to a depolarizing concentration of KCl (A) or stimulatory concentrations of glucose and other nutrient secretagogues (B).BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine then exposed for 1 h to the test agents. Cells were preincubated (1 h) in buffer containing 1.1 mmol/l glucose and the insulinotropic actions of 30 mmol/l KCl, 16.7 mmol/l glucose, 10 mmol/l mannose or 30 mmol/l 2-ketoisocaproic acid (KIC) were tested during a 60 min incubation. Fig (A) Value are mean ± SEM (n=6). ***p<0.001 compared with effects in the absence of addition (None). Fig (B) Value are mean ± SEM (n=6). *p<0.05 compared with effects of standard control culture.

  Figure 3. Effects of prolonged exposure to alanine on insulin secretion in response to a depolarizing concentration of KCl (A) or stimulatory concentrations of glucose and other nutrient secretagogues (B).BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine then exposed for 1 h to the test agents. Cells were preincubated (1 h) in buffer containing 1.1 mmol/l glucose and the insulinotropic actions of 30 mmol/l KCl, 16.7 mmol/l glucose, 10 mmol/l mannose or 30 mmol/l 2-ketoisocaproic acid (KIC) were tested during a 60 min incubation. Fig (A) Value are mean ± SEM (n=6). ***p<0.001 compared with effects in the absence of addition (None). Fig (B) Value are mean ± SEM (n=6). *p<0.05 compared with effects of standard control culture.

Figure 4 .

 4 Figure 4. Effects of prolonged exposure to either 10 mmol/l alanine or 10 mmol/l 2-aminoisobutyric (AIB) on subsequent insulin responses to amino acid test agents. BRIN-BD11 cells were cultured for 18 h in the absence (standard culture medium) or presence of 10 mmol/l alanine or 10 mmol/l AIB then exposed for 1 h to the test agents. Cells were preincubated (1 h) in buffer containing 1.1 mmol/l glucose and the insulinotropic actions of 10 mmol/l alanine, 10 mmol/l AIB or 10 mmol/l serine were tested during a 60 min incubation. Value are mean ± SEM (n=6). *p<0.05, ***p<0.001 compared with effects in the absence of addition (None). ∆ p<0.05, ∆∆ p<0.01, ∆∆∆ p<0.001 compared with effects after standard culture.

Figure 5 .

 5 Figure 5. (A) Effects of prolonged exposure to alanine on alanine-induced changes in membrane potential in BRIN-BD11 cells at 1.1 mmol/l glucose. Each data point represents mean ± SEM (n=6).Area-under-the-curves (AUCs) for standard culture or alanine culture were 6704000 ± 388324 and 5077334 ± 304649, respectively. (B) Effects of prolonged exposure to alanine on alanine-induced changes in intacellular Ca 2+ in BRIN-BD11 cells at 1.1 mmol/l glucose. Each data point represents mean ± SEM (n=6). Area-under-the-curves (AUCs) for standard culture or alanine culture were 573527 ± 28720 and 374636 ± 47437, respectively.

ClinicalFigure 6 .

 6 Figure 6. (A) 13 C NMR spectra of extracts of BRIN-BD11 cells incubated with 10 mmol/l L-[3-13 C]alanine for 1 h following alanine culture (i) compared with standard culture (ii). (B) The amount of glutamate and aspartate specifically labelled during 1 h incubation with L-[3-13 C]alanine after alanine culture or standard (control) culture. (C) Percentage enrichments of glutamate C2, C3 and C4 during 1 h incubation with L-[3-13 C]alanine after alanine culture or standard (control) culture. Values are mean ± SEM (n=3). *p<0.05 compared with control.

Figure 7 .

 7 Figure 7. Effects of prolonged exposure to alanine on PDK-2 protein and gene expression. BRIN-BD11 cells were cultured in the absence or presence of 10 mmol/l alanine and additionally exposed for 1 h to 10 mmol/l alanine before analyses. (A) Western blot analysis of PDK-2 protein expression. (B) Characterization of PDK-2/JNK ratio to parallel Western blot analysis. (C) Characterization of PDK-2/18s mRNA levels (arbitrary units) detected by semi quantitative reverse-transcription-PCR. Values are mean ± SEM (n=5) of target protein or gene over housekeeping protein (JNK) or gene (18 S). *p<0.05 compared with standard culture (control).

Figure 8 .A 1 Clinical 2 ClinicalFigure 4 Clinical

 8124 Figure 8. Effects of prolonged exposure to alanine on PDK-4 protein and gene expression. BRIN-BD11 cells were cultured in the absence or presence of 10 mmol/l alanine and additionally exposed for 1 h to 10 mmol/l alanine before analyses. (A) Characterization of PDK-4/GAPDH ratio by Western blot analysis. (B) Characterization of PDK-4/18s mRNA levels (arbitrary units) detected by semi quantitative reverse-transcription-PCR. Values are mean ± SEM (n=5) of target protein or gene over housekeeping protein (GAPDH) or gene (18 S). *p<0.05, **p<0.01 compared with standard culture (control).

Figure 6 Clinical

 6 Figure 6

  Figure 7

Glu and Asp labelled from [3-13 C]Alanine (nmol/ mg protein) Enrichments of glutamate C2, C3 and C4 (%)

	A	\ \	B
				Control	Alanine culture
			Glu C2	8.5 1.6	11.1 1.0
			Glu C3	7.3 1.1	8.2 0.6
			Glu C4	23.0 2.2	37.1 1.8 *
			Asp C2	10.0 0.8	8.6 0.9
			Asp C3	10.0 1.0	9.4 0.8
			C
				Control	Alanine culture
			Glu C2	12.9 1.1	13.2 0.5
			Glu C3	11.2 0.4	9.8 0.6
	Chemical Shift (ppm)		Glu C4	35.8 1.5	44.4 2.5 *

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgments

LB acknowledges the Health Research Board and UCD for funding. The authors also acknowledge support from the R & D Office of NI HPSS. Clinical Science Immediate Publication. Published on 14 Aug 2008 as manuscript CS20080138 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080138 A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Dr Jeremy F L Cobbold
email: j.cobbold@imperial.ac.uk

Quentin M Anstee

Robert D Goldin

Horace R T Williams

Helen C Matthews

Bernard V North

Nathan Absalom

Howard C Thomas

Mark R Thursz

Roger D Cox

S D Taylor-Robinson

I J Cox

Robert Steiner

PHENOTYPING MURINE MODELS OF NON-ALCOHOLIC FATTY LIVER DISEASE THROUGH METABOLIC PROFILING OF INTACT LIVER TISSUE

Keywords: Steatohepatitis, NAFLD, spectroscopy, NMR, Diabetes

Introduction

Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of liver disease encompassing steatosis (fatty change), non-alcoholic steatohepatitis (NASH) and cirrhosis in the absence of alcohol abuse [START_REF] Anstee | Mouse models in non-alcoholic fatty liver disease and steatohepatitis research[END_REF]. Population studies show that NAFLD is strongly associated with insulin resistance, type II diabetes mellitus, hypertension, obesity and dyslipidaemia [START_REF] Marchesini | Association of nonalcoholic fatty liver disease with insulin resistance[END_REF][START_REF] Powell | The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years[END_REF][START_REF] Sanyal | AGA technical review on nonalcoholic fatty liver disease[END_REF]. The pathogenic mechanisms remain poorly understood and are difficult to study in humans [START_REF] Koteish | Animal models of steatohepatitis[END_REF]. While an estimated 10-24% of adults worldwide have a degree of hepatic steatosis [START_REF] Angulo | Nonalcoholic fatty liver disease[END_REF], only a small proportion of these go on to develop progressive disease, characterised histologically by steatohepatitis with fibrosis. In order to target therapy to the appropriate patients there is a need for accurate, well-validated, methods to analyse hepatic lipid content and metabolism.

Magnetic resonance spectroscopy (MRS) provides information about the biochemical composition of tissues both in vivo and in vitro. In vivo, the technique has been used to assess the prevalence of NAFLD [START_REF] Szczepaniak | Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population[END_REF]; recently, more detailed information on lipid composition has been reported [START_REF] Johnson | Noninvasive assessment of hepatic lipid composition: Advancing understanding and management of fatty liver disorders[END_REF]. In vitro, MRS has been applied to the study of liver tissue extracts in obese rats [START_REF] Serkova | Metabolic profiling of livers and blood from obese Zucker rats[END_REF] and urine and plasma from mice with features of NAFLD [10;11]. In vitro proton magic angle spinning ( 1 H MAS) MRS allows simultaneous analysis of lipid and aqueous compartments of intact solid tissue, resembling in vivo findings more closely than tissue extracts. In addition, 1 H MAS MRS requires a smaller mass of tissue for analysis compared to extracts [9;12], and enables histological evaluation of the sample following analysis [START_REF] Dave | In vitro 1H-magnetic resonance spectroscopy of Barrett's esophageal mucosa using magic angle spinning techniques[END_REF]. 1 H MAS MRS has been applied to metabolic profiling in liver and other tissues from animal models and from humans [10;12;14-17].

Much of our understanding of the pathogenesis of NALFD is based on research using animal models [START_REF] Anstee | Mouse models in non-alcoholic fatty liver disease and steatohepatitis research[END_REF]. No existing model exhibits the entire NAFLD phenotype. Many differ pathologically from human disease in all but superficial histological appearance. Thus, research is hampered by the lack of reliable models of progressive steatohepatitis that accurately reflect the human disease. Currently, the phenotyping of such models is reliant on physical observation, indirect biochemical assay and histopathology, all of which are time-consuming. In addition, subtle changes in metabolism and hepatic disease severity may not be detected using conventional techniques. Liver histology may be used to document overt features of NAFLD, such as the presence of hepatocellular lipid droplets, inflammation and fibrosis. Histological assessment is semiquantitative and involves a degree of subjectivity in interpretation. There is a requirement for robust techniques to phenotype rapidly and categorise the output of programs that develop new models of NAFLD. In particular, there is a need to identify animals which exhibit the characteristics of more advanced disease, including steatohepatitis. Accordingly, the aims of this study were:

1. To characterise murine models of NAFLD using 1 H MAS MRS of intact liver tissue. 2. To assess the predictive value of multivariate models to determine the strain and diet for each mouse on the basis of 1 H MAS MRS differences. 3. To compare MR spectral differences between histologically characterised normal liver, steatosis and steatohepatitis.

Materials and Methods:

Animals

The study was approved by the local ethics committee and performed in accordance with the Animal (Scientific Procedures) Act 1986. Mice were housed under standard conditions (12 hour light/dark cycle, temperature 21±2°C, humidity 55±10%) and provided with food and access to drinking water ad libitum. Two inbred strains of mice, known to differ in susceptibility to dietary induced NAFLD, C3H/HeH (sensitive) and BALB/cAnNCrl (resistant) were used in these studies (each group, n = 6) [START_REF] Fearnside | Phylometabonomic patterns of adaptation to high fat diet feeding in inbred mice[END_REF]. In addition, a recently described genetic model of NASH maintained on a C3H/HeH background, Gena/263 "Buttermouse", was studied (each group, n = 6-10). The Gena/263 model was identified using a phenotype-driven n-ethyl-n-nitrosurea (ENU) mutagenesis screen for impaired glucose tolerance and histological/biochemical evidence of steatohepatitis [START_REF] Matthews | Buttermouse:the effect of high fat feeding on a new mouse model of insulin resistance and steatohepatitis[END_REF]. The liver was excised and half was snap-frozen in liquid nitrogen prior to storage at -80ºC, while the remainder was placed in 10% formalin. Formalin-fixed tissue was processed into paraffin wax and the sections were stained with haematoxylin & eosin, prior to histological examination by a histopathologist blinded to the phenotypic data, using the well validated NASH Clinical Research Network semi-quantitative scoring system for cellular morphology, inflammation and fibrosis [START_REF] Kleiner | Design and validation of a histological scoring system for nonalcoholic fatty liver disease[END_REF].

4 Proton MAS MRS experiments were carried out using a MAS probe (Doty Scientific Inc.) interfaced with a JEOL Eclipse 500+ spectrometer (JEOL (UK) Ltd, Welwyn Garden City, UK) and an 11.75T superconducting magnet (Oxford Instruments, Oxford UK). Samples were spun at 4000Hz and kept at 4ºC to minimise sample degradation during data acquisition [START_REF] Martinez-Granados | Metabolite identification in human liver needle biopsies by high-resolution magic angle spinning 1H NMR spectroscopy[END_REF]. A pulse and collect sequence (repetition time (TR) 10s, acquisition time 2.2s, 32 data collects) was used to derive a fully relaxed spectrum without suppression of the water resonance, the "total signal spectrum". A pulse and collect with a water presaturation sequence (TR 5s, acquisition time 2.2s, 64 data collects) provided water suppression, producing a spectrum dominated by lipid. A Carr-Purcell-Meiboom-Gill (CPMG) spin-echo pulse sequence with water presaturation (echo time 40ms, TR 5s, acquisition time 2.2s, 128 data collects) was employed to suppress macromolecular signal (mainly lipid) and emphasize lower molecular weight metabolite signals. In particular, this enabled the resolution and quantitation of the resonances within the total choline region. 32K data points were acquired for each spectrum with a spectral width of 15kHz [START_REF] Mahon | 1)H magnetic resonance spectroscopy of preinvasive and invasive cervical cancer: in vivo-ex vivo profiles and effect of tumor load[END_REF]. The overall time for data acquisition was 35 minutes.

Data analysis and interpretation

Spectra were processed using KnowItAll ® Informatics System v7.8 (Bio-Rad, Philadelphia, USA). Free induction decays were zero-filled by a factor of 2 and multiplied by an exponential windows function with a 1Hz line-broadening function prior to Fourier transformation. Spectral resonances were assigned according to the published literature [12;14-16]. Analysis was divided into hypothesis-generating multivariate analysis and biologically relevant conventional analysis of specific metabolites.

The spectral range of δ0-10.0ppm was analysed, including the water region for the pulse and collect sequences and excluding the region containing the residual water resonance (δ4.8-5.2ppm) in the sequences with water-presaturation. Spectra were scaled to the sum of the total spectral integral (water included for sequences without water suppression and excluded for those with water presaturation) to enable relative quantitative analysis of metabolites between samples.

Multivariate data analysis

The 1 H MAS MR spectrum from intact liver tissue demonstrated numerous identifiable resonances or peaks corresponding to specific metabolites (Figure 1). Accordingly, multivariate data-reduction techniques were applied in order to use information from the whole spectrum, and to determine which peaks contributed to the metabolic variation between individual samples and between groups of samples.

MR spectra acquired using the pulse and collect sequence with water presaturation were divided into small regions ("buckets") of 0.04±0.02ppm, representing specific metabolite functional groups, placed using an "intelligent bucketing" algorithm in the Knowitall ® software. The integral of each bucket was then quantified and expressed as a proportion of the total spectral integral.

Unsupervised multivariate factor analysis was performed using principal components analysis (PCA) to assess clustering and the presence of outliers. PCA is an unbiased A c c e p t e d M a n u s c r i p t technique, which is independent of any classifying information (such as strain) and the output summarises the metabolite variation within the dataset as a whole. Each principal component represents a combination of peaks, representing metabolites, expressed as a vector. The principal components are combined orthogonally, avoiding duplication or redundancy of information. Scores plots show the position of the samples in the newly defined multidimensional space, where the axes represent each orthogonal principal component. Examination of the loadings plots allows identification of those metabolites which predominantly account for variability within the dataset.

Supervised analysis was performed using partial least squares discriminant analysis (PLS-DA) to investigate factors associated with class membership, with the Pirouette v4.0 software package (Infometrics, Washington, USA). Here, the classifying information is included in the analysis, so the output summarises the MR spectral (metabolic) differences between the groups. Orthogonal signal correction (OSC) is a filtering technique applied to PLS-DA analyses to remove variation in the spectra, which is unrelated to the defined conditions [START_REF] Gavaghan | Physiological variation in metabolic phenotyping and functional genomic studies: use of orthogonal signal correction and PLS-DA[END_REF]. To determine the predictive power of the model, cross-validation was performed by leaving out each sample in turn (a leave-one-out algorithm), and a model was constructed from the remaining samples to predict the class membership of the excluded sample. Such an algorithm makes best use of a small dataset, where a separate validation dataset is not possible. The Q 2 value indicates the validity of the discrimination, with values >0.05 considered significant [22;23].

Examination of spectral resonances

In order to examine the specific differences between metabolite ratios of prior interest and those identified in the loadings plots, peak integrals (proportional to the total quantity of the metabolite in the sample) were measured. The lipid:water ratio was derived from the pulse and collect spectra without water suppression, both as the sum of the integrals of all the fatty acid proton resonances and as the methylene proton:water peak expressed as a percentage. The polyunsaturated to monounsaturated fatty acid ratio index (PUFA:MUFA index) was derived from the pulse and collect spectra with water suppression, and comprised the resonance at δ2.80ppm (defined as the diallylic bond protons found in polyunsaturated fatty acids and denoted as "PUFA") divided by the resonance at δ5.33ppm (-CH=CH-functional group, found in mono-and polyunsaturated fatty acids, denoted as "MUFA"). The polyunsaturation index (PUI) was defined as the diallylic resonance divided by the sum of the allylic, diallylic, methyl and methylene resonances, expressed as a percentage [START_REF] Johnson | Noninvasive assessment of hepatic lipid composition: Advancing understanding and management of fatty liver disorders[END_REF]. Total choline (tCho) was derived from the pulse and collect spectra with water suppression and comprised the summed integral of the choline region resonances from δ3.18-δ3.28ppm, expressed as a percentage of the total spectral integral. The tCho region was further analysed using the CPMG data. Specifically, the resonance at δ3.26ppm, assigned as phosphatidylcholine (PtC) including contributions from betaine and trimethylamine-N-oxide (TMAO) and denoted as "PtC", was compared to the tCho levels.

Significant differences in phenotypic data were sought by ANOVA testing with post-hoc Bonferroni analysis. Glucose Tolerance ANOVA analysis identified significant differences in blood glucose levels at all IPGTT time points (Table 1 Glucose and glycogen resonances were also prominent. A number of amino acid resonances were also visible. The sum of the integrals for all lipid peaks was compared to the integral of the methylene lipid peak in all samples, demonstrating strong correlation between measures (Spearman rho correlation, r=0.998, p<0.0005). Accordingly, the integral of the methylene proton resonance was considered a surrogate for total lipid when describing lipid quantities relative to water.

Principal components analysis scores plots (data not shown) demonstrated clustering and separation of each mouse model from its control. This included the separation of models on the basis of diet (CD or HFD), strain (BALB/c and C3H, both on CD and HFD) and also presence of the Gena263 mutation from the wildtype control, both on CD and HFD.

Supervised PLS-DA models were then constructed. Correct class prediction was achieved with 100% accuracy for 6 of 7 comparisons of differing diet, strain and presence of the 263 mutation (Table 2).

Metabolites contributing to the spectral regions correlating strongly (positively or negatively) with each model compared to its control are documented in Table 3. Lipid moieties, choline-related compounds, glucose, glycogen and a number of amino acid resonances (including glutamine, leucine, lysine and alanine) were found to contribute to class separation. In particular, PtC (with contributions from betaine and TMAO) was found to be a major contributor to the PLS-DA models. Accordingly, PtC was integrated using the CPMG spectra and divided by the tCho region for comparison with histological class. The use of tCho as a denominator allowed relative quantitation of PtC without the potentially confounding dilutional effect of large changes in total lipid.

In addition to the separation of each model from its control, PLS-DA models were constructed to enable the classification of a single sample from the total dataset. 54 samples were included in the total dataset and class membership established with 100% accuracy in all but one model (Table 4).

Comparison between MAS MRS and histological assessment.

The distinction between simple steatosis and steatohepatitis is of importance for the development of models, which reflect progressive disease in humans. Therefore PCA was performed on spectra from mice of all strains fed HFD and stratified by histological assessment of NAFLD. The samples were divided into histologically normal, steatotic and steatohepatitic groups on the basis of histological assessment of an adjacent slice of tissue. Steatohepatitis was defined in this study as the presence of hepatocyte ballooning +/-the presence of necroinflammatory infiltrate. All samples in the steatohepatitis group had evidence of hepatocyte ballooning. PCA demonstrated clustering of the groups, so PLS-DA was performed to assess the factors associated with category membership. Category membership (between histologically normal, steatotic and steatohepatitic samples) using the leave-one-out algorithm was predicted with very high accuracy, as shown in Table 5.

The intrahepatic lipid level (median, interquartile range) differed significantly between samples histologically categorised as normal (1.96%, 1.02), steatosis (8.09%, 3.71) and steatohepatitis (10.56%, 6.08) (Figure 3A). There was a significant decrease in the PUFA:MUFA index between the normal (0.68, 0.09) and either steatotic (0.37, 0.12) or steatohepatitic (0.32, 0.09) samples. between the steatotic and steatohepatitic groups (Figure 3B). The PUI was decreased in steatotic (3.40%, 0.95) and steatohepatitic mice (2.95%, 1.13) compared to normal mice (6.29%, 1.25) (Mann-Whitney U test, p<0.001). Therefore, the increase in PUFA:MUFA index can be interpreted as consequent to a systematic decrease in PUFA with increasing disease severity. The resonances at δ3.26ppm (PtC with TMAO and betaine) and δ3.22-3.23ppm (PC and GPC) were found consistently to contribute to the difference between mouse classes on multivariate statistical modelling. tCho was significantly decreased in steatohepatitis (0.31%, 0.19) compared to steatosis (0.49%, 0.28) (p=0.01) and in both compared to the histologically normal samples (1.61%, 0.69) (p<0.005) (Figure 3C). Furthermore, PtC/tCho was significantly lower in the steatotic (33.7%, 16.8) and steatohepatitic animals (27.6%, 3.68) compared to normal animals (45.5%, 6.14) (Figure 3D). These findings are consistent with reports of low plasma PtC and high urinary choline oxidation products seen in 129S6 mice fed HFD [START_REF] Dumas | Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice[END_REF].

Relationship between intrahepatic lipid and obesity in murine models

Mouse mass plotted against the %lipid:water (Figure 4A) demonstrated that intrahepatic lipid increased exponentially with increasing mouse mass at time of culling (R 2 = 0.80, p<0.0005). The %lipid:water was also compared to the histolological assessment of steatosis and demonstrated a close relationship between steatosis score and %lipid:water (Figure 4B).

Discussion

These data demonstrate that metabolic profiling of intact liver tissue using 1 H MAS MRS combined with PLS-DA is a powerful technique for the discrimination of NAFLD in animal models, differentiating by genetic (strain and the Gena/263 mutation), environmental (dietary) and histological factors. In particular, the first evidence is presented of accurate discrimination of histologically-defined steatosis and steatohepatitis using MRS of intact tissue. 1 H MAS MRS with PLS-DA also enables the separation and classification of different mouse strains on differing diets, despite normal histological appearances. Furthermore, it has been established that an exponential relationship exists between hepatic steatosis and obesity in these murine models.

In liver tissue, the "total signal" MR spectra are dominated by water and lipid resonances. Such spectra allow the quantitation of lipid, either in absolute terms, or as a proportion of the total signal in the spectrum. This marker of hepatic steatosis is a quantitative objective linear variable which compares favourably with the semi-quantitative subjective histological assessment of steatosis (Figures 4B) [START_REF] Szczepaniak | Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population[END_REF]. This is an important observation as steatosis is prerequisite for the development of steatohepatitis and consequent fibrotic liver disease. In HFD-fed animals, there was a progressive significant increase in the [%lipid:water] from normal through steatotic to steatohepatitic samples. This may represent increased uptake of fatty acids by the liver, decreased export or decreased catabolism [START_REF] Anstee | Mouse models in non-alcoholic fatty liver disease and steatohepatitis research[END_REF]. Additionally, defective peripheral fatty acid uptake shunts more fatty acids to the liver [START_REF] Goldberg | Ins and outs modulating hepatic triglyceride and development of nonalcoholic fatty liver disease[END_REF]. Intrahepatic lipid increased exponentially with animal mass, suggesting that fatty acid uptake peripherally was maximised in these mice at a weight approaching 40g. Thereafter, intrahepatic lipid accumulated in preference to peripheral uptake of fatty acids and deposition of triacylglycerol (TAG). Accumulated free fatty acids may be disposed of by esterification to triglycerides and by fatty acid oxidation producing oxidative stress and initiating inflammation [START_REF] Yamaguchi | Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis[END_REF]. While the traditional two-hit hypothesis proposed that the development of steatosis is followed by a second hit initiating inflammation [START_REF] Day | Steatohepatitis: a tale of two "hits[END_REF], recent work by Yamaguchi and colleagues suggests that this model is now too simple, and that visible lipid deposition is less pathogenically relevant [START_REF] Yamaguchi | Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis[END_REF]. Differential fatty acid partitioning between esterification, leading to the accumulation of visible fat and oxidation of the invisible FFA, is more relevant to the pathogenesis. The use of MRS techniques in this study enables the determination of total lipid composition, which includes the assessment of the invisible hepatic FFA component.

The relative decrease in PUFA seen with increasing disease severity supports previous evidence, that a decrease in PUFAs has been observed in in the liver and blood of obese Zucker rats [START_REF] Serkova | Metabolic profiling of livers and blood from obese Zucker rats[END_REF] and in the serum of human subjects with hepatic steatosis and steatohepatitis [27;28]. A reduced PUFA:MUFA ratio has been considered to represent a surrogate marker of lipid peroxidation, associated with oxidative stress [START_REF] Serkova | Metabolic profiling of livers and blood from obese Zucker rats[END_REF]. Depletion of long chain PUFA (LC-PUFA) within the liver is thought to increase FA and TAG synthesis by upregulation of lipogenic genes via reduced downregulation of sterol regulatory binding protein (SREBP-1), and to decrease FA oxidation, including by a reduction of PPAR-α activation [START_REF] Videla | Oxidative stress and depletion of hepatic long-chain polyunsaturated fatty acids may contribute to nonalcoholic fatty liver disease[END_REF].

PtC is an essential phospholipid [START_REF] Li | Phosphatidylcholine and choline homeostasis[END_REF], existing as a membrane-bound macromolecule giving rise to a broad resonance [START_REF] Millis | Classification of human liposarcoma and lipoma using ex vivo proton NMR spectroscopy[END_REF] within which the -N(CH 3 ) 3 functional group of trimethylamine-N-oxide (TMAO) and betaine (oxidation products of choline metabolism) are co-resonant [START_REF] Rooney | High-resolution diffusion and relaxation-edited magic angle spinning 1H NMR spectroscopy of intact liver tissue[END_REF]. Measurement of choline levels demonstrates a decrease in cholinecontaining products in the obese models, and in those animals with steatosis and 10 A c c e p t e d M a n u s c r i p t steatohepatitis, compared to histologically normal controls. Importantly, the tCho is significantly lower in steatohepatitis compared to simple steatosis. This is consistent with the observation that mice fed a choline-deficient diet develop hepatic steatosis and steatohepatitis [START_REF] Walkey | Biochemical and evolutionary significance of phospholipid methylation[END_REF]. However, increasing lipid, which dominates the spectrum, may also contribute to the reduction in tCho observed, by means of a dilution effect. Therefore, the PtC resonance (also containing betaine and TMAO) was integrated in isolation and expressed as a ratio of the total choline region. PtC biosynthesis from choline and methionine is required for normal hepatic secretion of VLDL [START_REF] Yao | The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes[END_REF], and decreased PtC leads to accumulation of intracellular triglyceride [START_REF] Noga | An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins[END_REF]. Indeed, when comparing the PtC resonance to the total choline region, PtC is reduced compared to other cholinecontaining products in mice with hepatic steatosis and steatohepatitis compared to histologically normal livers. This is consistent with work on aqueous liver tissue extract in obese Zucker rats: a reduction in betaine at δ3.26 compared to lean controls [START_REF] Serkova | Metabolic profiling of livers and blood from obese Zucker rats[END_REF] has been shown to protect against alcoholic hepatic steatosis in experimental animals [START_REF] Kharbanda | Betaine attenuates alcoholic steatosis by restoring phosphatidylcholine generation via the phosphatidylethanolamine methyltransferase pathway[END_REF].

As with any technique, there are limitations to this approach to studying NALFD. While 1 H MAS MRS allows the quantitation of protons within functional groups of metabolites, it is not possible directly to quantify specific fatty acids, as this requires accurate measurement of fatty acyl chain length and the position and number of functional groups within the chain. Nevertheless, the extent of saturation or (poly)unsaturation is of biological and clinical relevance. The overlap of certain resonances (particularly small resonances near the larger lipid peaks) may reduce the accuracy of integration of those peaks. However, by dividing the spectra into small regions or "buckets", quantitation is objective and consistent. Curve-fitting software is used in the in vivo setting and enables quantitation of small numbers of overlapping peaks. Newer approaches such as "targeted profiling" may improve quantitation of the multiple peaks found in complex tissue and biofluid samples in vitro [START_REF] Weljie | Targeted profiling: quantitative analysis of 1H NMR metabolomics data[END_REF].

This study has demonstrated that a small number of resonances contribute reliably to the differentiating the cohorts studied. Despite low field strength and the presence of motion artefact, tCho and a number of lipid resonances may be resolved in human liver in vivo [START_REF] Fischbach | Assessment of in vivo 1H magnetic resonance spectroscopy in the liver: a review[END_REF], raising the possibility that 1 H MRS could provide discriminatory information in the context of NAFLD in vivo.

In conclusion, this study has demonstrated the utility of 1 H MAS MRS of intact liver tissue in conjunction with multivariate modelling techniques for the investigation and classification of murine models of NAFLD on the basis of environmental, genetic and histopathological features. Class membership may be predicted with high accuracy and it is possible to distinguish reliably between samples, even where histological appearances are indistinguishable. PLS-DA enables the identification of biologicallyrelevant metabolites contributing to the differences between models. Future studies may employ these techniques to screen the output of programs generating murine models of NAFLD and other liver diseases. Greater subject numbers will facilitate the use of separate large training and test sets. High resolution 1 H MAS MRS data may be used to aid the interpretation of future non-invasive in vivo MRS studies. As the resolution of 1 H MR spectra from in vivo MR scanners improves, combination with multivariate statistical analysis may allow detailed non-invasive assessment of hepatic disease in animals and humans.

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  Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159 A c c e p t e d M a n u s c r i p t

  The PUFA:MUFA index did not differentiate 8 Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159 A c c e p t e d M a n u s c r i p t

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  Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159

Clinical

  Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159 A c c e p t e d M a n u s c r i p t 12 Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. 13 Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1.

Figure 1 .

 1 Figure 1.Water-suppressed, fully relaxed 1 H MAS-MR spectrum from a C3H mouse on CD. The residual water resonance at δ4.8-5.2 was removed as indicated. Key: L1, lipid CH 3 CH 2 (methyl); L2 lipid (CH 2 ) n (methylene); L3 lipid CH 2 CH 2 CO (methylene to L5); L4 lipid CH=CHCH 2 CH 2 (allylic); L5 lipid CH 2 CH 2 CO (α-methylene to carboxyl); L6 lipid CH=CHCH 2 CH=CH (diallylic-"PUFA"); L7 lipid CH=CH (methene-"MUFA"); Ala, alanine; Cho, choline; PC, phosphocholine; GPC, glycerophosphocholine; PtC, phosphatidylcholine; TMAO, trimethylamine-N-oxide; Glc, glucose; Glyc, glycogen; Urd, uridine; Tyr, tyrosine.

Figure 2 17 Clinical

 217 Figure 2.

Figure 2 .

 2 Figure 2. Figure shows representative 1 H MAS MRS spectra without and with water suppression, with paired histology from samples with normal architecture (A, A' and B), steatosis (C, C' and D) and steatohepatitis (E, E' and F). The "total signal" MR spectra (A, C, E) illustrate the ratio of water to other metabolites. The "lipid signal" MR spectra (A', C', E') amplify other major metabolite groups. The lipid consituents increase, while the tCho decreases with increasing disease severity. Characteristic histological features of steatosis (lipid droplets) and steatohepatitis (hepatocyte ballooning) from the corresponding tissue sample are shown in D and F.

Figure 3 Figure 3 .

 33 Figure 3.

  Figure 4.

Figure 4 .

 4 Figure 4. Scatter graphs showing the lipid:water ratio plotted against A) the mass of mouse in grams and B) the histological steatosis score.

Table 1 )

 1 . Gena/263 mice exhibited mild spontaneous steatosis and a more moderate steatosis with some ballooning degeneration when fed HFD. A small but significant increase in serum ALT was observed between C3H dietary groups (HFD 47.60±4.41 versus CD 30.20±2.54 IU/L, p=0.009) and between HFD-fed C3H and BALB/c mice (47.60±4.41 versus 17.00±1.78 IU/L, p=0.001).

	MR Characterisation
	A representative water-supressed 1 H MAS-MR spectrum is shown in Figure 1 and
	illustrates the specific, quantifiable lipid resonances, including the fatty acid functional
	groups. The choline region (δ3.18-δ3.28ppm) contains four clearly identifiable peaks
	representing	choline	(δ3.20ppm),	phosphocholine	(PC)	(δ3.22ppm),
	glycerophosphocholine (GPC) (δ3.23ppm) and PtC with contributions from TMAO and
	betaine at δ3.26ppm.

). HFD-fed C3H mice had significantly higher mean t=120 minute blood glucose concentrations than BALB/c mice fed HFD (11.86±0.96 versus 7.30±1.80 mmol/l, t-test, p<0.001). A trend towards impaired glucose tolerance was observed between HFD and CD-fed C3H animals but this did not reach statistical significance (11.86±0.96 versus 10.07±0.41 mmol/l). HFD fed Gena/263 mutant animals had significantly higher t=120 blood sugar levels than all other groups (p<0.001) including HFD-C3H mice (20.68±0.53 versus 11.86±0.96 mmol/l).

Histology

In keeping with the blunted weight gain and preservation of glucose tolerance in response to high fat diet, BALB/c mice were highly resistant to dietary induction of steatosis showing no histological evidence of steatosis in either CD or HFD groups at 36 weeks post-randomisation. In contrast, HFD fed C3H animals consistently exhibited a florid macro and micro-vesicular steatosis (median steatosis score 2/3) associated with ballooning hepatocyte degeneration (score 1/2) and a mild necroinflammatory infiltrate (score 1/3) (

Table 1 .

 1 Phenotype of mice and effects of the high fat diet (HFD).

			Mean Body Weight /g (SEM) †	Mean IPGTT at 12 Weeks	Median	NAS	Hist.
						/mmol/L (SEM)		Score*
			6 Weeks 20	36	T=0	T=60	T=120	St.	Fib. Inf. Ball.
				Weeks	Weeks
	C3H	Control 25.38	35.32	43.31	6.83	15.38	10.07	0	0	0	0
			(0.59)	(2.58)	(2.58)	(0.22)	(1.94)	(0.41)
		HFD	25.24	44.72	55.24	10.42	21.26	11.86	2	0	1	1
			(0.23)	(0.34)	(0.80)	(0.53)	(1.17)	(0.96)
	BALB/c	Control 24.29	26.49	31.40	4.47	16.13	7.66	0	0	0	0
			(0.83)	(0.87)	(1.53)	(0.99)	(2.48)	(1.52)
		HFD	23.25	27.52	35.29	3.58	11.34	7.30	0	0	0	0
			(0.89)	(0.70)	(1.11)	(0.20)	(2.87)	(1.80)
			6

Weeks 20 Weeks 36 Weeks Mean IPGTT at 12 Weeks /mmol/L (SEM)

									Median	NAS	Hist.
									Score*
						T=0	T=60	T=120	St.	Fib. Inf. Ball.
	Gena/263 Control 24.56	41.60	-	10.84	26.66	22.43	1	0	0	2
			(1.2) ‡	(2.06)		(1.81)	(2.13)	(1.27)
		HFD	25.38	45.50	-	9.31	28.65	20.68	3	0	0	1
			(0.46)	(0.64)		(0.95)	(1.42)	(0.53)
	Non-Mut	Control 27.89	42.40	-	5.22	11.67	8.15	0	0	0	0
			(0.5) ‡ -	(2.57)		(1.93)	(0.29)	(1.14)
		HFD	26.58	44.76	-	8.42	23.43	13.30	2	0	0	1
			(0.33)	(0.79)		(0.65)	(0.86)	(0.36)

Table 1 .

 1 Phenotype of mice and effects of the high fat diet (HFD) on body mass, glucose tolerance and histological scoring are tabulated. † Weight recorded at start of experiment (age 6 weeks), mid-experiment and at cull point. BALB/c and C3H were culled at 36 weeks, Gena/263 mice (and C3H controls) were culled at 20 weeks. ‡ 6 week control diet data from separate cohort of animals to those used in MRS study, shown for illustrative purposes. * NAS Score: Steatosis (0-3), Fibrosis (0-4), Inflammation (0-3), Ballooning Hepatocyte degeneration (0-2). 21 Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080159 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080159

	M a n u s c r i p t
	A c c e p t e d

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements J.F.L.C holds the Centenary Fellowship from the Hammersmith Hospitals Trustees Research Committee. Q.M.A. is a Medical Research Council funded Clinical Research Fellow. H.R.T.W is funded by the Broad Medical Research Program, USA. H.C.M holds a Research Training Fellowship from the Wellcome Trust. H.C.T, S.D.T-R, M.R.T and I.J.C are grateful for support from the NIHR Biomedical Research Centre funding scheme.

Table 2. A comparison of each model against its control in dataset. PLS-DA models were constructed to separate the MR spectra obtained from a given mouse model from its control as specified, with one orthogonal factor removed (OSC).