Ee L Lim

Kieren G Hollingsworth

Fiona E Smith

Peter E Thelwall

Roy Taylor
email: roy.taylor@ncl.ac.uk

Inhibition of lipolysis in type 2 diabetes normalizes glucose disposal without change in muscle glycogen synthesis rates

Keywords: glucose disposal, lipolysis, magnetic resonance spectroscopy, muscle glycogen, non-esterified fatty acid, type 2 diabetes APE, atom percentage excess, CV, coefficient of variation, MR, magnetic resonance, MRS, MR spectroscopy, NEFA, non-esterified fatty acid

Suppression of lipolysis by acipimox is known to improve insulin-stimulated glucose disposal and this is an important phenomenon. The mechanism has been assumed to be enhancement of glucose storage as glycogen, but no direct measurement has tested this concept nor its possible relationship to the reported impairment in insulin-stimulated muscle ATP production. Isoglycaemic-hyperinsulinaemic clamps with 13 C-glucose infusion were performed on type 2 diabetes subjects and matched controls with measurement of glycogen synthesis by 13 C magnetic resonance spectroscopy (MRS) of muscle. 31 P saturation transfer MRS was used to quantify muscle ATP turnover rates. Glucose disposal rates were restored to near-normal in diabetic subjects after acipimox (6.2 ± 0.8 vs. 4.8 ± 0.6 mg/kg ffm /min; P<0.01; control 6.6 ± 0.5 mg/kg ffm /min). The increment in muscle glycogen concentration was two-fold higher in controls compared with the diabetic group and acipimox administration to the diabetic group did not increase this (2.0 ± 0.8 vs. 1.9 ± 1.1 mmol/l; P<0.05; control 4.0 ± 0.8 mmol/l). ATP turnover rates did not increase during insulin stimulation in any group, but a modest decrease in the diabetes group was prevented by lowering plasma NEFA (8.4 ± 0.7 vs. 7.1 ± 0.5 mol/g/min; P<0.05; controls 8.6 ± 0.8 mol/g/min). Suppression of lipolysis increases whole-body glucose uptake with no increase in the rate of glucose storage as glycogen but with increase in whole-body glucose oxidation rate. ATP turnover rate in muscle exhibits no relationship to the acute metabolic effect of insulin.

INTRODUCTION

Suppression of lipolysis by acipimox or nicotinic acid has long been known to improve meal tolerance and insulin sensitivity [START_REF] Fulcher | Metabolic effects of suppressions of non-esterified fatty acid levels with acipimox in obese NIDDM subjects[END_REF][START_REF] Vaag | Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus[END_REF]. Sustained suppression of plasma non-esterified fatty acids (NEFA) is associated with a decrease in intra-muscular long-chain fatty acyl-CoAs and it is believed that this enhances glucose metabolism via substrate competition [START_REF] Bajaj | Effect of a sustained reduction in plasma free fatty acid concentration on intramuscular long-chain fatty Acyl-CoAs and insulin action in type 2 diabetic patients[END_REF]. The mechanism for this interaction is well described, involving control of cellular glucose uptake in muscle [START_REF] Roden | Mechanism of free fatty acid-induced insulin resistance in humans[END_REF]. Both glucose oxidation and non-oxidative metabolism are involved, and as one of the original descriptions reported increase in the active form of glycogen synthase it has been assumed that increased rates of glucose storage as glycogen underlie the phenomenon [START_REF] Vaag | Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus[END_REF]. However, there is no direct in vivo evidence that glycogen synthesis rate is enhanced by NEFA suppression. This mechanism is important in fully understanding the insulin resistance of type 2 diabetes and evaluating potential interventions.

Recently, demonstration of subnormal insulin stimulation of ATP turnover rates in type 2 diabetes has been postulated as a basic defect which limits glucose metabolism [START_REF] Kelley | Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes[END_REF][START_REF] Ritov | Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes[END_REF][START_REF] Szendroedi | Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes[END_REF][START_REF] Petersen | Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes[END_REF]. It is not known whether such a defect underlies change in glucose uptake or glycogen synthesis itself. By employing 31 P magnetic resonance spectroscopy (MRS) together with 13 C spectroscopy to track glycogen metabolism it is possible to examine this fundamental problem. In this study, we tested the hypothesis that acute suppression of lipolysis would improve the rate of glucose metabolism specifically by increasing muscle glycogen synthesis. The effect of decreasing fatty acid availability upon muscle mitochondrial ATP turnover rates was also examined.

MATERIALS AND METHODS Subjects

Ten subjects with well-controlled type 2 diabetes (three females and seven males) and eight physical activity-, age-and BMI-matched normal glucose tolerant subjects (two females and six males) were studied (Table 1). All subjects were recruited by means of advertisement and underwent a complete medical history, clinical examination, and lab tests to exclude hepatic and renal diseases. Patients with diabetes on insulin treatment or any oral hypoglycaemic medications apart from metformin were excluded. The individuals in the control group had neither a family history of diabetes nor were taking any medication known to affect glucose tolerance or insulin sensitivity. All subjects with diabetes and no control subjects were taking statins. Normal glucose metabolism was confirmed by a standard oral 75g glucose tolerance test. None of the subjects performed moderate or intense exercise on a regular basis. Physical activity was assessed over 3 days using the Body Monitoring System and SenseWear Armband (BodyMedia®, Pittsburgh, USA) which provides a measure of total daily energy expenditure and number of steps taken per day [START_REF] Mignault | Evaluation of the Portable HealthWear Armband: a device to measure total daily energy expenditure in free-living type 2 diabetic individuals[END_REF]. The research was carried out in accordance with the Declaration of Helsinki (2000) and the study protocol was approved by the Newcastle upon Tyne Ethics Committee No. 2. Informed consent was obtained from all subjects.

Experimental protocol

All subjects refrained from planned physical exertion during the 3 days preceding the studies and fasted overnight for 12 hours before the experiments. Metformin was withdrawn 3 days before each experiment. Type 2 diabetes subjects were each studied on two days, 4-8 weeks apart. Doses of acipimox (250mg) were given at baseline and 3 hours later, or on the placebo test day, identical placebo tablets were given in a double blind fashion. The body weight and lifestyle of the subjects remained unchanged during the period of the study. Control subjects were studied on one day only with administration of placebo tablets.

Isoglycaemic-hyperinsulinaemic clamps

For all experiments, subjects travelled to the magnetic resonance centre by taxi and were transported within the centre by wheelchair. At 0830 hrs (-270 min), one cannula was inserted into an antecubital vein for administration of glucose and insulin. A second cannula was inserted into the contralateral wrist vein for blood sampling. Use of a hand warming device ensured arterialization of venous blood. Isoglycaemia was maintained in order to ensure that the true basal condition of each participant could be observed and the entire study was carried out with the subject lying in the MR scanner. Isoglycaemic hyperinsulinaemia was maintained for 150 min using the clamp technique [START_REF] Defronzo | Glucose clamp technique: a method for quantifying insulin secretion and resistance[END_REF]. Insulin (Actrapid; NovoNordisk, Bagsvaerd, Denmark) was administered as a primed-continuous infusion (40 mU/m 2 /min). To inhibit pancreatic hormone secretion, somatostatin was infused at 0.06 g/kg/min (Somatostatin-UCB; UCB-Pharma, Netherlands) from 5 min before the start of the insulin infusion and continued for the duration of the clamp. In order to increase sensitivity of measurement of the glycogen synthesis rate by 13 C MRS, the variable glucose infusion contained 20% [1-13 C]-glucose (Cambridge Isotope Laboratories, Andover, MA). Wholebody insulin sensitivity was determined from calculated whole-body glucose disposal during the last 30 min of the hyperinsulinaemic glucose clamp [START_REF] Defronzo | Glucose clamp technique: a method for quantifying insulin secretion and resistance[END_REF]. Whole-body glucose disposal was calculated from glucose infusion rate [START_REF] Rizza | Dose-response characteristics for effects of insulin on production and utilization of glucose in man[END_REF]. To assess rate of oxidation of infused glucose, breath samples for 13 CO 2 measurement were obtained just before the start of the clamp (0 min), at 15 min, 45 min, 90 min and at the end of the clamp (150 min). As this measure would be affected by differences in plasma glucose enrichment, the index of whole body glucose oxidation was calculated as the ratio of breath to plasma 13 C APE ([breath APE/plasma APE]x100).

MRS examinations

Magnetic resonance data were acquired using a 3T Achieva scanner (Philips, Best, The Netherlands) with an in-built body coil used for imaging. A 14-cm diameter surface coil was used for phosphorus spectroscopy and a 6-cm diameter 13 C coil with an integral quad 1 H decoupling coil (PulseTeq, Wotton under Edge, UK) was used for 13 C spectroscopy. Subjects remained supine inside the magnetic resonance spectrometer with each coil positioned beneath the widest part of the left calf during each investigation. The coil position was marked on the leg with indelible ink. Scout images were acquired to ensure identical coil positioning on repeat scans. To prevent movement during each study, the coil was secured in place using webbing straps around the calf. Analysis of all spectra was performed with jMRUI (version 3.0) [START_REF] Naressi | Java-based graphical user interface for MRUI, a software package for quantitation of in vivo/medical magnetic resonance spectroscopy signals[END_REF] using the AMARES fitting algorithm [START_REF] Vanhamme | Time-domain quantification of series of biomedical magnetic resonance spectroscopy signals[END_REF].

decoupled 13 C pulse-acquire sequence (TR = 200 ms, spectral width = 8 kHz, 3000 averages, WALTZ decoupling, nominal tip angle = 80) over a 15-min acquisition time. Calibration of 13 C spectra was performed by comparison of in vivo glycogen [1-13 C] signal amplitudes with that of a standard glycogen solution (100 mM glycogen, 70 mM KCl, 0.05% sodium azide). Quantitation was performed by comparison of signal amplitude to those from a leg-shaped phantom acquired at a range of separations between coil and phantom, representative of the separation between coil and muscle due to skin and subcutaneous fat. The same coils, pulse sequences, and tip angles as employed for in vivo spectra were used for acquisition of these calibration data. 13 C MRS spectra were acquired from subjects at baseline from -205 to -185 min and from -25 to -5 min and twice further during the isoglycaemic-hyperinsulinaemic clamp from 50-70 min and from 130-150 min. The concentration of muscle glycogen at baseline, [Glyc] muscle , was calculated using the formula:

phantom phantom muscle muscle [Glyc] [Glyc] S S =
where S phantom and S muscle are the signal intensities arising from glycogen in the phantom and muscle, respectively, and [Glyc] phantom is the concentration of glycogen in the phantom (100 mmol/l). The increments in muscle glycogen concentration at 70 min and 150 min of the clamp, [ΔGlyc 70 ] and [ΔGlyc 150 ] respectively, were calculated from the equation previously reported [START_REF] Jue | Direct observation of glycogen synthesis in human muscle with 13C NMR[END_REF]:

[DGlyc 70 ] = (S 70 -S 0 ) ´[Glyc 0 ]´f 0 S 0 ´f 70 [DGlyc 150 ] = (S 150 -S 70 )
´[Glyc 0 ]´f 0 S 0 ´f150 where S 0 , S 70 and S 150 represent the signal intensity of 13 C-glycogen at 0, 70 and 150 min, [Glyc 0 ] is the basal glycogen concentration in mmol/l. f 0 corresponds to the natural abundance enrichment of 13 C-glycogen at baseline (1.1%), and f 70 and f 150 correspond to the mean percentage 13 C enrichment of plasma glucose at 70 min and 150 min respectively. Each increment was then added to the previous concentration, and the slope calculated by linear regression analysis to yield the rate of glycogen synthesis [START_REF] Shulman | Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy[END_REF].

Analytical techniques

Plasma glucose concentration was measured by the glucose oxidase method (YSI glucose analyzer; Yellow Springs, OH). The 13 C enrichment in plasma glucose was determined by gas chromatography-mass spectrometry of the pentaacetate derivatives of plasma glucose after deproteinization and deionization as previously described [START_REF] Wolfe | Tracers in metabolic research: radioisotope and stable isotope/mass spectrometry methods[END_REF]. Plasma insulin levels were determined using ELISA kits (DAKO, Ely, UK). Plasma NEFA concentration was measured on a Roche Cobas centrifugal analyzer using a Wako kit (Wako Chemicals, Nuess, Germany).

Breath 13 C enrichments

Breath samples for 13 C enrichments were collected at 5 time points during the study. The subjects were asked to exhale fully through a short straw into a glass tube (Labco Exetainer; Laboco Ltd., Buckinghamshire, UK). The tube was immediately stoppered. 13 C enrichment of breath samples was determined by continuous flow isotope ratio mass spectrometry (ABCA system; PDZ Europa). The coefficient of variation (CV) for the analysis was 0.07% and CV for the collection was 0.3%. All results of the 13 C enrichment of expired air measurements are expressed as atom percentage excess (APE).

Statistical analysis

Data are presented as means ± SE, unless otherwise stated. Statistical analyses were performed using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). Statistical comparisons between diabetic and control groups were performed using Student's t test while within group differences were determined using paired t test where appropriate. Changes of sequential data within experiments were evaluated by repeated measures ANOVA with post hoc Tukey correction where appropriate. Statistical significance was accepted at P<0.05.

RESULTS

Plasma glucose, insulin and NEFA

The experimental conditions necessary to test the hypothesis were achieved. During the 3 hour baseline observation, plasma glucose concentration remained steady in the control subjects (5.1 ± 0.1 to 5.0 ± 0.1 mmol/l) and gradually fell as expected in the diabetic subjects (7.7 ± 0.3 to 6.5 ± 0.3 mmol/l; P=0.002). As per protocol, plasma glucose concentration was clamped at 5.0 ± 0.1 mmol/l in control group, 6.6  0.2 mmol/l in diabetes/placebo group and 6.4 ± 0.2 mmol/l in diabetes/acipimox group respectively (P<0.001 diabetes vs. control; Figure 1A). Clamp plasma insulin concentrations were stable at 573 ± 18 pmol/l (Figure 1B). Plasma NEFA concentrations rapidly suppressed below 0.2 mmol/l in the diabetes/acipimox study (P=0.005) and were stable and similar in diabetes/placebo and control groups until commencement of insulin infusion (Figure 1C).

Glucose disposal rates and muscle glycogen concentrations

During the clamp, glucose disposal rate was lower in the diabetes/placebo group compared with the control group (4.8 ± 0.6 vs. 6.6 ± 0.5 mg/kg ffm /min, P=0.04; Figure 2A). Prior suppression of plasma NEFA by acipimox brought about a 23% increase of glucose disposal rate in the type 2 diabetic group (6.2 ± 0.8 vs. 4.8 ± 0.6 mg/kg ffm /min, acipimox vs. placebo respectively, P=0.005; Figure 2A) so that it approached that of the control group (6.6 ± 0.5 mg/kg ffm /min; P=0.72). Fasting muscle glycogen concentration was similar in all three groups (diabetes/placebo: 67.5 ± 4.5 mmol/l, diabetes/acipimox: 69.0 ± 2.1 mmol/l and control: 71.1 ± 2.6 mmol/l) and remained unchanged during the basal period. The increment in muscle glycogen concentration after 70 min of insulin stimulation was small and not significantly different between the three groups. By the end of the clamp, insulin-stimulated muscle glycogen concentration increased by +4.0 ± 0.8 mmol/l in the control group and +1.9 ± 1.0 mmol/l in the diabetes/placebo group (P<0.05; Figure 2B). Prior administration of acipimox did not change the increment in muscle glycogen (+2.0 ± 0.8 mmol/l; Figure 2B). The mean rate of muscle glycogen synthesis from 70 to 150 min was 40 ± 7 μmol/l/min in the control subjects, 19 ± 9 μmol/l/min in the diabetes/placebo and 22 ± 7 μmol/l/min in the diabetes/acipimox groups (P<0.05 for control vs. both diabetes groups).

Breath 13 C enrichments 13 C APE in expired breath increased during the clamp in all groups (control 0.23 ± 0.04 and 0.44 ± 0.05; diabetes/placebo 0.14 ± 0.01 and 0.31 ± 0.03; diabetes/acipimox 0.22 ± 0.02 and 0.44 ± 0.03 at 90 and 150 min respectively). In order to compare rates of glucose oxidation corrected for plasma glucose enrichment the ratio of breath to plasma 13 C APE was examined. At the end of the clamp period this index of glucose oxidation was 2.53±0.17 vs. 3.16±0.21 for diabetes/placebo and diabetes/acipimox respectively (p<0.005) and 2.87±0.24 for controls (Figure 2C)

Muscle ATP turnover rate

Baseline ATP turnover rate in the diabetes/placebo group (8.6 ± 0.8 mol/g/min of muscle) was similar to that of the control subjects (8.6 ± 0.7 mol/g/min of muscle; Figure 3). Muscle ATP turnover rate in the control group did not change during the basal period or during the insulin infusion (end of clamp: 8.6 ± 1.3 mol/g/min of muscle). A gradual decline in ATP turnover rate was observed during the course of the diabetes/placebo group. This decline was not seen in the diabetes/acipimox group such that by the end of the clamp, ATP turnover rate 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.

was significantly higher than in the diabetes/placebo group (8.4 ± 0.7 vs. 7.1 ± 0.5 mol/g/min of muscle; p=0.02).

DISCUSSION

This study demonstrates that prior suppression of lipolysis effectively normalizes glucose infusion rate during hyperinsulinaemia in type 2 diabetes, but has no effect upon the rate of muscle glycogen synthesis. In contrast, the subnormal rate of whole-body glucose oxidation in type 2 diabetes was increase. The muscle ATP turnover rate was found to be normal in type 2 diabetes but could be modulated to a degree by suppression of lipolysis.

In type 2 diabetes, insulin-stimulated whole-body glucose uptake was observed to be associated with a 2-fold lower incorporation of 13 C glucose into glycogen. Acipimox brought about a 23% increase in glucose infusion rate during hyperinsulinaemia, similar to that reported by others [START_REF] Vaag | Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus[END_REF][START_REF] Bajaj | Effect of a sustained reduction in plasma free fatty acid concentration on intramuscular long-chain fatty Acyl-CoAs and insulin action in type 2 diabetic patients[END_REF]. Previously observed increases in glucose disposal rates during acipimox administration have been attributed to change in rate of glycogen synthesis [START_REF] Vaag | Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus[END_REF][START_REF] Lindegaard | Inhibition of lipolysis stimulates peripheral glucose uptake but has no effect on endogenous glucose production in HIV lipodystrophy[END_REF]. Clearly this was not the case over the 6 hours of NEFA suppression in the present study, a period which has been shown to be sufficient for the effect upon glucose metabolism to be maximal [START_REF] Roden | Mechanism of free fatty acid-induced insulin resistance in humans[END_REF]. The concept that the synthesis rate of glycogen would be increased under such circumstances derives from observation of an increase in the active form of muscle glycogen synthase, even though no change in muscle glycogen concentrations could be detected [START_REF] Vaag | Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus[END_REF]. Subsequently it has been demonstrated that change in activation state of glycogen synthase by suppression of lipolysis occurs in the absence of change in the proximal steps of insulin activation of glycogen synthase (GSK-3β and Akt Thr308) [START_REF] Lindegaard | Inhibition of lipolysis stimulates peripheral glucose uptake but has no effect on endogenous glucose production in HIV lipodystrophy[END_REF], and its relevance to substrate flux must be questioned.

Glucose infusion rate is a useful independent measure of overall glucose disposal, but does not take into account possible incomplete suppression of hepatic glucose production during the clamp. An estimate of endogenous glucose production can be obtained from the measured isotopic enrichments of infusate and plasma together with glucose infusion rate [START_REF] Ravikumar | Pioglitazone decreases fasting and postprandial endogenous glucose production in proportion to decrease in hepatic triglyceride content[END_REF]. Hepatic glucose production was 0.01, 1.13 and 0.39 mg/kg ffm /min in control, diabetes/placebo and diabetes/acipimox groups. By adding this value to the glucose infusion rate, total glucose disposal rates can be estimated to be 6.61, 5.93 and 6.59 mg/kg ffm /min respectively. Hence, acipimox brought about an 11% increase in total glucose disposal. Isoglycaemic hyperinsulinaemic clamps were used in this study in order to observe the physiological mechanisms operating at ambient blood glucose concentrations in the diabetic and control subjects, and in respect of the primary comparison between acipimox and placebo studies in the diabetic subjects clamp glucose levels were similar.

Whole-body glucose oxidation rate was increased approximately 25% by acipimox administration. This could not be quantified using indirect calorimetry in the present study as subjects remained in the 3T magnet throughout the test periods. However, the extent of acipimox-induced change in glucose oxidation has previously been shown to be approximately one third of the simultaneous change in rate of whole body glucose uptake [START_REF] Vaag | Effect of the antilipolytic nicotinic acid analogue acipimox on whole-body and skeletal muscle glucose metabolism in patients with non-insulin-dependent diabetes mellitus[END_REF]. It is likely that processes additional to glucose oxidation and muscle glycogen synthesis could explain the change in whole body glucose uptake. Glycolysis with lactate release (Cori cycle activity) could account for some of the change. Approximately 15% of a glucose load is taken up by skeletal muscle then released as lactate in normal health [START_REF] Kelley | Skeletal muscle glycolysis, oxidation, and storage of an oral glucose load[END_REF] and accounts for approximately 45% of systemic lactate appearance [START_REF] Consoli | Skeletal muscle is a major site of lactate uptake and release during hyperinsulinemia[END_REF]. In type 2 diabetes, the proportion of glucose taken up by muscle then released as lactate is 2 to 3-fold higher than normal during hyperinsulinaemia [START_REF] Mitrakou | of abnormal muscle and liver glucose metabolism to postprandial hyperglycemia in NIDDM[END_REF][START_REF] Capaldo | Glucose and gluconeogenic substrate exchange by the forearm skeletal muscle in hyperglycemic and insulin-treated type II diabetic patients[END_REF]. This flux could be affected if early steps in glucose metabolism were rate limiting so that the increased transport of glucose into the muscle cell by low fatty acid availability caused increased export from muscle as lactate, and an 18% increase in plasma lactate has been observed to accompany acipimox induced suppression of lipolysis during a clamp [START_REF] Vaag | Defective insulin activation of glycogen synthase in skeletal muscle in first degree relatives to patients with type 2 (non-insulin dependent) diabetes mellitus[END_REF]. Further work is required to examine specifically the acute changes in glycolysis and glucose oxidation in response to change in NEFA supply.

Although the change in plasma NEFA is a useful general indicator of rate of lipolysis, suppression of lipolysis would be expected to exert greatest effect upon muscle metabolism by preventing breakdown of intra-myocellular triglyceride and decreasing intracellular availability of fatty acid and diacylglycerols. Evidence is accumulating for diacylglycerols to be major modulators of substrate level control of metabolism [START_REF] Chibalin | Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance[END_REF][START_REF] Erion | Diacylglycerol-mediated insulin resistance[END_REF]. In the present study the overweight controls subjects were not restudied with acipimox, and although it would appear likely that similar effects would be produced, this awaits experimental verification.

This study observed no significant abnormality in muscle ATP turnover rate in overnight fasting type 2 diabetes subjects in comparison with exercise-, weight-, age-and sex-matched non-diabetic subjects, and this contrasts with reports of decreased basal mitochondrial function in type 2 diabetes [START_REF] Schrauwen-Hinderling | Impaired in vivo mitochondrial function but similar intramyocellular lipid content in patients with type 2 diabetes mellitus and BMI-matched control subjects[END_REF][START_REF] Phielix | Lower intrinsic ADP-stimulated mitochondrial respiration underlies in vivo mitochondrial dysfunction in muscle of male type 2 diabetic patients[END_REF]. Matching for daily physical activity may be of importance, as the greatest deficits in ATP turnover rates have been reported in a study which explicitly compared extremes of insulin sensitivity with no matching for habitual physical activity [START_REF] Petersen | Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes[END_REF]. Both basal ATP turnover rates and the independently assessed phosphocreatine recovery times have been reported to be normal in type 2 diabetes when compared with exercise matched controls [START_REF] Trenell | Increased daily walking improves lipid oxidation without changes in mitochondrial function in Type 2 diabetes[END_REF][START_REF] De Feyter | Early or advanced stage type 2 diabetes is not accompanied by in vivo skeletal muscle mitochondrial dysfunction[END_REF]. However, the major reason for the apparent discrepancy is clear from the work of Schrauwen-Hinderling who demonstrated that in vivo basal mitochondrial function was related to fasting blood glucose, with normal values for diabetic subjects at levels similar to those reported here [START_REF] Schrauwen-Hinderling | Impaired in vivo mitochondrial function but similar intramyocellular lipid content in patients with type 2 diabetes mellitus and BMI-matched control subjects[END_REF][START_REF] Phielix | Lower intrinsic ADP-stimulated mitochondrial respiration underlies in vivo mitochondrial dysfunction in muscle of male type 2 diabetic patients[END_REF]. More recent work from the same group has demonstrated that change in metabolic state alters measured mitochondrial function [START_REF] Hoeks | Prolonged fasting identifies skeletal muscle mitochondrial dysfunction as consequence rather than cause of human insulin resistance[END_REF]. In the type 2 diabetes subjects given placebo, a fall in muscle ATP turnover rate was observed during the fasting fall in plasma glucose. It is possible that the downward trend occurred with increasing duration of fasting and consequent increased mobilization of fatty acids from intramyocellular lipid (IMCL) as acipimox administration prevented the fall in ATP turnover rate and would have inhibited intracellular lipolysis. This phenomenon requires further study.

The present study was designed in the expectation of inducing an increase in ATP turnover rate with insulin in the non-diabetic control subjects as it was believed that increase in ATP turnover would precede or accompany the acute changes in cellular metabolism. We observed no effect of insulin upon ATP turnover rates. However, the previously reported insulin effect on mitochondrial function related to measurements averaged over 150 to 350 minutes of insulin stimulation [START_REF] Szendroedi | Muscle mitochondrial ATP synthesis and glucose transport/phosphorylation in type 2 diabetes[END_REF][START_REF] Petersen | Decreased insulin-stimulated ATP synthesis and phosphate transport in muscle of insulin-resistant offspring of type 2 diabetic parents[END_REF][START_REF] Brehm | Increased lipid availability impairs insulin-stimulated ATP synthesis in human skeletal muscle[END_REF][START_REF] Brehm | Acute elevation of plasma lipids does not affect ATP synthesis in human skeletal muscle[END_REF]. The method of quantifying ATP turnover used in the present study was optimized so that measurements could be carried out frequently over 30 minute periods for the first time. When it became clear that there was no stimulation of ATP turnover rates in the matched control subjects of the present study, a separate investigation of time course of stimulation of muscle ATP turnover was conducted and has been published [START_REF] Lim | Measuring the acute effect of insulin infusion on ATP turnover rate in human skeletal muscle using phosphorus-31 magnetic resonance saturation transfer spectroscopy[END_REF]. We observed no change in ATP turnover rate during the first 45 minutes of insulin infusion when the change in glucose metabolism was maximal, yet in young, insulin sensitive subjects there was a slow steady increase evident after 2 hours. This sub-study concluded that the acute metabolic effect of insulin does not depend upon measureable increases in ATP turnover rate. Processes separate from the insulin effect on glucose metabolism, such as the insulin effect on mitochondrial fusion and proliferation [START_REF] Molina | Mitochondrial networking protects beta-cells from nutrient-induced apoptosis[END_REF][START_REF] Yang | Insulin stimulates Akt translocation to mitochondria: implications on dysregulation of mitochondrial oxidative phosphorylation in diabetic myocardium[END_REF] or mitochondrial protein synthesis [START_REF] Stump | Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts[END_REF], may affect ATP turnover rate on a timescale of several hours of insulin stimulation. These processes are temporally dissociated from and not relevant to the early metabolic effects of insulin and led to the belief that insulin acutely changes ATP turnover. However, suppression of plasma NEFA in the type 2 diabetes subjects during the 5.5 hour protocol did modestly affect the rate of ATP turnover, and the converse finding of elevation of plasma NEFA and suppression of ATP turnover has previously been reported [START_REF] Brehm | Increased lipid availability impairs insulin-stimulated ATP synthesis in human skeletal muscle[END_REF]. Other recent work has demonstrated decreased glucose disposal during short-term elevation of plasma NEFA without change in ATP turnover rate in healthy subjects [START_REF] Brehm | Acute elevation of plasma lipids does not affect ATP synthesis in human skeletal muscle[END_REF] and the time course of change in fatty acid supply requires further study. Overall, change in metabolic state rather than diabetes per se can affect measured ATP turnover rates in muscle.

As all subjects with type 2 diabetes were taking statin therapy, reflecting current clinical practice, the possible effect of this on glucose metabolism must be considered. Statins do not affect glucose tolerance nor modulate 72 hour glucose profiles, but may bring about a very small beneficial effect on insulin sensitivity as shown in a randomized crossover study designed for this purpose [START_REF] Huptas | Effect of atorvastatin (10 mg/day) on glucose metabolism in patients with the metabolic syndrome[END_REF]. No impact of statins would be expected on the primary comparison of acipimox or placebo exposure within the diabetic group.

The present data demonstrate that lowering fatty acid availability increased whole body glucose uptake without increasing the rate of glucose storage as muscle glycogen but with increase in whole-body glucose oxidation rates. A change in fasting ATP turnover rate in muscle was observed after suppression of lipolysis in type 2 diabetes, but this change was associated with no effect on muscle glycogen synthesis. A c c e p t e d M a n u s c r i p t

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Legends to Figures

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Clinical

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Clinical

  Science Immediate Publication. Published on 10 Mar 2011 as manuscript CS20100611 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100611

Clinical

  Science Immediate Publication. Published on 10 Mar 2011 as manuscript CS20100611 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100611A 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.

Clinical

  Science Immediate Publication. Published on 10 Mar 2011 as manuscript CS20100611 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100611 A c c e p t e d M a n u s c r i p t

  Figure 1. Time course of plasma glucose (A), insulin (B) and NEFA (C) concentrations during the isoglycaemic-hyperinsulinaemic clamps in control (○), diabetes/placebo (Δ) and diabetes/acipimox (▲) studies. Data are means ± SE. *P<0.05 diabetes vs. control.

Figure 2 .

 2 Figure 2. Upper panel: Glucose infusion rates during the final 30 minutes in the isoglycaemic-hyperinsulinaemic clamps: control, diabetic with placebo and diabetic with acipimox. Mid panel: Incremental changes in muscle glycogen concentration in the three studies. Lower panel: Ratio of breath 13 CO2 to plasma 13 C-glucose APE at the end of the each study. *P<0.05; **P<0.005.

Figure 3 . 2 Clinical

 32 Figure 3. ATP turnover rate in control (open bar), diabetic with placebo (hatched bar) and diabetic with acipimox (filled bar) during basal period and after 120 min of isoglycaemichyperinsulinaemia. *P<0.05.

Table 1

 1 Characteristics of subjects (means ± SE)

	T2DM	Control	P
	(7M, 3F)	(6M, 2F)

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Table 2

 2 Plasma enrichment of 13C-glucose (APE) Clinical Science Immediate Publication. Published on 10 Mar 2011 as manuscript CS20100611 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100611

	Time	0	15	45	90	150
	Control	0	3.79±0.37	7.50±0.72	12.31±0.66	15.15±0.70
	D/Placebo	0	2.99±0.44	6.34±0.50	9.65±0.60	12.16±0.57
	D/Acipimox	0	3.81±0.40	7.99±0.45	11.33±0.60	14.18±0.63

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

P MRS was carried out as previously described[START_REF] Lim | Measuring the acute effect of insulin infusion on ATP turnover rate in human skeletal muscle using phosphorus-31 magnetic resonance saturation transfer spectroscopy[END_REF] and acquired at baseline from -240 to -210 min and from -60 to -30 min and twice further during the isoglycaemic-hyperglycaemic clamp from 15-45 min and from 90-120 min. For 13 C MRS, the distance between the 13 C/ 1 H decoupling coil surface and the muscle was recorded from scout images. The 13 C pulse power was calibrated to a nominal value of 80 in the tissue of interest by observing the powerdependent variation in signal from a fiducial marker located in the coil housing, containing a sample exhibiting a[START_REF] Vanhamme | Time-domain quantification of series of biomedical magnetic resonance spectroscopy signals[END_REF] C signal with short T 1 (213 mM [2-13 C]-acetone and 25 mM GdCl 3 in water). Spectra showing the glycogen [1-13 C] resonance were acquired using a non-localized 1 H-

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

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. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGEMENTS

We are most grateful to the volunteers for their commitment during the study. We thank Mrs Jean Gerrard and Ms Mei Jun Chen, senior research nurses for their assistance during the clamps, Ms Louis Morris and Mrs Carol Smith, senior radiographers and Mrs Heather Cook and Mrs Annette Lane for laboratory assistance. The work was funded by Wellcome Trust [grant number 073561] and the Newcastle upon Tyne MRC Biomedical Research Centre. Clinical Science Immediate Publication. Published on 10 Mar 2011 as manuscript CS20100611 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100611

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.

AUTHOR CONTRIBUTIONS

E.L. researched data, contributed to discussion and wrote manuscript. K.H. researched data, contributed to discussion and reviewed/edited manuscript. F.S. researched data and reviewed/edited manuscript. P.T. contributed to discussion and reviewed/edited manuscript. R.T. researched data, contributed discussion and reviewed/edited manuscript.


Saad Al-Kaddah

Katrin Reder-Christ

Gabriela Klocek

Imke Wiede- Mann

Manuela Brunschweiger

Gerd Bendas
email: gbendas@uni-bonn.de

Imke Wiedemann

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

Keywords: Antibiotics, Gallidermin, Isothermal Titration Calorimetry (ITC), Lipid II, Surface Acoustic Wave (SAW) biosensor, Vancomycin, AMPs, antimicrobial peptides, DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol, ITC, Isothermal Titration Calorimetry, MC 540, merocyanine 540, MIC, minimal inhibitory concentration, QCM, Quartz Crystal Microbalance, SAW, Surface Acoustic Wave sensor

The interaction of the lantibiotic gallidermin and the glycopeptide antibiotic vancomycin with bacterial membranes was simulated using mass sensitive biosensors and isothermal titration calorimetry (ITC). Both peptides interfere with cell wall biosynthesis by targeting the cell wall precursor lipid II, but differ clearly in their antibiotic activity against individual bacterial strains. We determined binding affinities of vancomycin and gallidermin to model membranes ± lipid II in detail. Both peptides bind to DOPC/lipid II membranes with high affinity (K D 0.30 µM and 0.27 µM). Gallidermin displayed also strong affinity to pure DOPC membranes (0.53 µM) an effect that was supported by ITC measurements. A surface acoustic wave (SAW) sensor allowed measurements in the picomolar concentration range and revealed that gallidermin targets lipid II at an equimolar ratio and simultaneously inserts into the bilayer. These results indicate that gallidermin, in contrast to vancomycin, combines cell wall inhibiton and interference with the bacterial membrane integrity for potent antimicrobial activity.

Introduction

Several antibiotics display their activity by interacting with the bacterial membrane. Beside non-specific membrane disturbing activities, as described for many antimicrobial peptides (AMPs) [START_REF] Shai | Mode of action of membrane active antimicrobial peptides[END_REF][START_REF] Lohner | Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics[END_REF], target related modes of action are known. To date several antibiotic compounds were described, which bind to lipid anchored cell wall precursors, and thus inhibit cell wall biosynthesis. The first described example was vancomycin, a glycopeptide that targets a key molecule of peptidoglycan biosynthesis, the cell wall precursor lipid II [START_REF] Chatterjee | Compounds formed between nucleotides related to the biosynthesis of bacterial cell wall and vancomycin[END_REF][START_REF] Wong | Peptidoglycan biosynthesis. Unexploited antibacterial targets within a familiar pathway[END_REF][START_REF] Breukink | Lipid II as a target for antibiotics[END_REF]. Later, several lantibiotics, lanthionine containing peptides, were identified, which also target lipid II. In the case of nisin, the prototype of the type-A lantibiotics, the interaction with lipid II leads to a dual mode of action, i.e. cell wall biosynthesis is blocked, and in addition, lipid II is recruited as a pore constituent for permeabilizing the bacterial membrane [START_REF] Breukink | Lipid II is an intrinsic component of the pore induced by nisin in bacterial membranes[END_REF][START_REF] Brötz | Role of lipid-bound peptidoglycan precursors in the formation of pores by nisin, epidermin and other lantibiotics[END_REF][START_REF] Hasper | Assembly and stability of nisin-lipid II pores[END_REF][START_REF] Hsu | The nisin-lipid II complex reveals a pyrophosphate cage that provides a blueprint for novel antibiotics[END_REF][START_REF] Van Heusden | Lipid II induces a transmembrane orientation of the pore-forming peptide lantibiotic nisin[END_REF].

Gallidermin, a type-A lantibiotic produced by Staphylococcus gallinarum shows striking structural similarities to nisin in the N-terminal double ring system which had been identified as the binding motif for targeting the pyrophosphate moiety of lipid II [START_REF] Kellner | Gallidermin: a new lanthionine-containing polypeptide antibiotic[END_REF]. In contrast to nisin, due to the short molecular length, gallidermin is hardly able to form pores in membranes via peptide-lipid II complexes. Therefore, the primary mode of action of gallidermin is obviously based on the interaction with lipid II resulting in inhibition of cell wall synthesis [START_REF] Bonelli | Insights into in vivo activities of lantibiotics from gallidermin and epidermin mode-of-action studies[END_REF].

Nevertheless, although gallidermin and vancomycin target the same molecule in the bacterial membrane, striking differences in the antibiotic activity against some bacterial strains are observed. While the MICs of gallidermin and vancomycin against Staphylococcus simulans 22 are in the µM range, 0.15µM and 0.05µM, respectively, gallidermin is much more potent than vancomycin against e.g. Lactobacillus lactis HP and Lactobacillus fermentum ATCC 9338 (0.005 µM and 0.001 µM vs. 0.21 µM and >6.9 µM)

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ACCEPTED MANUSCRIPT (unpublished). These observation might be explained by the different membrane interactions of both peptides. Vancomycin targets the D-Ala-D-Ala motif of lipid II [START_REF] Sheldrick | Structure of vancomycin and its complex with acetyl-D-alanyl-D-alanine[END_REF][START_REF] Gilbert | Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction[END_REF] and has not been described to interact with bacterial membranes, which is in good agreement with the hydrophilic polar structure of the glycopeptide (Figure 1). In contrast, gallidermin comprises hydrophobic moieties which allow for membrane interaction. This is in line with our recent results showing that gallidermin strongly associates with model membranes [START_REF] Christ | Membrane lipids determine the antibiotic activity of the lantibiotic gallidermin[END_REF]. It might be anticipated that insertion of gallidermin into the bilayer has an impact on membrane order, and thus indirectly affects the vital function of lipid II and contributes to antimicrobial activity.

Here we compared gallidermin and vancomycin with respect to the membrane interaction and targeting of lipid II to get a deeper insight into gallidermin activities. The use of model membranes in combination with different analytical techniques allowed us to focus on peptide-membrane-interactions, as schematically depicted in Figure 1.

Cyclic voltammetry (data not presented) could exclude that both peptides affected the membrane barrier function. Kinetic binding constants of both peptides to DOPC ± lipid II model membranes were determined using a quartz crystal microbalance (QCM) and data from a surface acoustic wave sensor (SAW) allowed further insight into gallidermin membrane interactions at varying peptide/lipid ratios. These findings were further supported by isothermal titration calorimetry (ITC) to provide the thermodynamic profile of peptide-membrane interaction [START_REF] Cooper | Microcalorimetry of protein-protein interactions[END_REF][START_REF] Holdgate | Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics[END_REF]. ITC has been successfully applied in a number of studies to analyze the membrane interaction of different peptides and proteins [START_REF] Binder | Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes[END_REF][START_REF] Wenk | Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation[END_REF][START_REF] Rekharsky | Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall[END_REF][START_REF] Breukink | Binding of Nisin Z to bilayer vesicles as determined with isothermal titration calorimetry[END_REF].

However, to the best of our knowledge, ITC has neither been used to analyze the interaction of peptides with lipid II containing membranes nor gallidermin in general.

Our data indicate that gallidermin, in contrast to vancomycin, interacts strongly with model membranes and simultaneously targets lipid II nearly at a 1:1 binding ratio. This sheds

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light on the mode of action of gallidermin; especially on the processes at the bacterial membrane surface which result in effective killing.

Materials and Methods

Materials

1-hexadecanethiol (C 16 H 33 SH) was purchased from FLUKA (Neu-Ulm, Germany). 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3phosphoglycerol (DOPG), 11-amino-1-undecanethiol hydrochloride, merocyanine 540 (MC 540), and vancomycin were purchased from Sigma-Aldrich (München, Germany).

Gallidermin was purchased from Genmedics (Reutlingen, Germany), and C55P was from Larodan (Malmö, Sweden). Lipid II was synthesized using membrane preparations of M. luteus DSM 1790 [START_REF] Brötz | The lantibiotic mersacidin inhibits peptidoglycan biosynthesis at the level of transglycosylation[END_REF] and purified as described [START_REF] Schneider | In vitro assembly of a complete, pentaglycine interpeptide bridge containing cell wall precursor (lipid II-Gly5) of Staphylococcus aureus[END_REF].

Millipore ® water was taken from water Purification System Elix 5 (Millipore GmbH, Schwalbach, Germany). ITC buffers were prepared from 25 mM Tris, and 50 mM sodium chloride (Sigma Aldrich, München, Germany) at pH = 7.4. PBS (pH = 7.4) was prepared according to standard protocols.

QCM experiments

The modification of the quartz crystals has already been described [START_REF] Christ | The detection of UVinduced membrane damages by a combination of two biosensor techniques[END_REF][START_REF] Christ | The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques[END_REF]. A DOPC or DOPG ± 0.1 mol% lipid II layer was transformed by Langmuir-Blodgett technique onto a 1-hexadecanethiol covered quartz crystal. QCM experiments were performed at 25°C using a LiquiLab21 system (ifak e.V., Barleben, Germany). After reaching a constant frequency, 3 mL of a 1 µM peptide solution (gallidermin, and vancomycin, respectively)

were injected into the flow system. The run was stopped after reaching a constant frequency again. The binding constants were calculated from the frequency run as described [START_REF] Christ | Membrane lipids determine the antibiotic activity of the lantibiotic gallidermin[END_REF].

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Liposome preparation

Liposomes were prepared using the lipid hydration technique. First, a 30 mM aliquot of DOPC, dissolved in chloroform, was put in a 10 mL round-bottom flask. Chloroform was removed under vacuum by using a rotary evaporator resulting in a thin lipid film. Small glass pellets and 5 mL of Millipore ® water or the indicated buffers were added to the batch and vortexed. Spontaneously formed multilamellar vesicles were shaken overnight and extruded using a Lipex Basic Extruder (Northern Lipids, Vancouver, Canada) seven times through an Isopore ® polycarbonate filter with a pore size of 0.1 µm (Millipore GmbH, Schwalbach, Germany) resulting in large unilamellar vesicles (LUVs). Liposomal particle size was characterized by dynamic light scattering (Malvern autosizer IIc, Malvern, UK).

Phospholipid concentration was calculated according to the inorganic phosphate determination using ascorbate-molybdate mixture and colorimetric detection of the resulting dye at 812 nm, as described by Ames and Dubin [START_REF] Ames | The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid[END_REF].

SAW measurements

The S-Sens ® K 5 surface acoustic wave biosensor (SAW GmbH, Bonn, Germany) was Raw data were processed using the Origin ® software provided with the instrument. Blank values were determined in control titrations by injecting LUVs into the indicated solvents.

Although dilution heat was small and negligible, it was included in the final analysis.

Under assumption of an one-site binding model with the ligand in the sample cell, thermodynamic parameters as enthalpy (∆H), entropy (∆S) and the equilibrium binding constant (K D ) were calculated.

Merocyanine 540 assay

Merocyanine 540 (MC540) was added to the liposome batch from a stock solution (20 µg/mL PBS) to reach a final dye:lipid molar ratio of 1:100. MC 540 containing liposomes were incubated for 20 min at 8°C before peptides were added in a peptide:lipid ratio of 1:10 or 1:1, resp. The fluorescence was measured with a spectrofluorometer (Perkin Elmer LS1, Waltham, USA) at 584 nm and excitation wavelength at 540 nm.

Statistical methods

QCM experiments were repeated for at least four times. Kinetic binding constants are represented as mean value ± standard deviation. The statistical significance of the effects

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was determined by the unpaired Student`s t-test. A p value of ≤ 0.05 was considered for statistical significance. ITC experiments and SAW measurements were repeated for at least seven times, representative ITC scans and SAW curves were selected for presentation. The fluorescence measurements were repeated three times.

Results and Discussion

Kinetics of peptide binding to model membranes

Kinetic constants of peptide binding to model membranes give an insight into antibiotic activity referring to different modes of membrane interaction or entering the bacterial cell.

A QCM biosensor was applied to detect peptide binding kinetics of gallidermin and vancomycin. To consider the role of lipid II as molecular target and its impact on binding intensity, 0.1 mol% lipid II was added to the matrix lipids. In accordance to other peptide/model membrane studies [START_REF] Martínez | Specific interaction of the unmodified bacteriocin Lactococcin 972 with the cell wall precursor lipid II[END_REF][START_REF] Arnusch | Enhanced membrane pore formation by multimeric/oligomeric antimicrobial peptides[END_REF][START_REF] Wiedemann | Lipid IIbased antimicrobial activity of the lantibiotic plantaricin C[END_REF][START_REF] Ruhr | Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles[END_REF][START_REF] Böttiger | Influence of Ca(2+) ions on the activity of lantibiotics containing a mersacidin-like lipid II binding motif[END_REF][START_REF] Ghalit | Synthesis of bicyclic alkene-/alkane-bridged nisin mimics by ring-closing metathesis and their biochemical evaluation as lipid II binders: toward the design of potential novel antibiotics[END_REF][START_REF] Ringstad | An electrochemical study into the interaction between complementderived peptides and DOPC mono-and bilayers[END_REF] and to avoid non-specific binding of the cationic antimicrobial peptides to the membrane surface, DOPC was initially selected as membrane lipid. As depicted in Fig 2A, lipid II as the bacterial target is crucial for vancomycin membrane binding. Compared to pure DOPC membranes, vancomycin binding to DOPC/lipid II is nearly tenfold stronger (0.30 µM vs. 2.42 µM). Gallidermin displays a comparable high affinity binding in presence of lipid II, but a much higher affinity to pure DOPC membranes (0.53 µM) than vancomycin. This suggests to differences in the mode of membrane interaction of both peptides, independent of lipid II targeting. While vancomycin membrane interaction have not been reported [START_REF] Beauregard | Dimerization and membrane anchors in extracellular targeting of vancomycin group antibiotics[END_REF][START_REF] Domenech | Interactions of oritavancin, a new lipoglycopeptide derived from vancomycin, with phospholipid bilayers: Effect on membrane permeability and nanoscale lipid membrane organization[END_REF], the gallidermin structure (Fig. 1) give clear indication for membrane interaction forced by the hydrophobic moieties.

With respect to the net negative charge of bacterial membranes, the twofold positive charge of gallidermin should also have an impact on the non-targeted membrane affinity.

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In order to simulate this, gallidermin binding affinies to DOPC/DOPG (9:1) membranes were detected (Fig. 2B). Membrane charge, which is assumed to induce the inital electrostatic attraction of the peptide to the membrane, is of minor effect in this approach.

Although the affinities of gallidermin to the charged membranes are slightly higher than to the neutral ones, the differences between pure and lipid II containing membranes in turn are marginal (0.22 µM vs. 0.11 µM, resp.). This indicates that membrane charge only plays a minor role in membrane affinity of gallidermin.

To evaluate the binding process of both peptides to the DOPC membranes in more detail, association and dissociation rate of vancomycin were determined as depicted in Fig. 2C and D in comparison to the recently published gallidermin binding data [START_REF] Christ | Membrane lipids determine the antibiotic activity of the lantibiotic gallidermin[END_REF]. Lipid II induces a faster association rate of vancomycin (Fig. 2C) and a decelerated dissociation (Fig. 2D) than on pure DOPC. In contrast to vancomycin, gallidermin binding is differently affected by lipid II. Gallidermin has more than a twofold higher association rate to pure DOPC membranes than in presence of lipid II, which is threefold higher than for vancomycin (Fig. 2C). This dominantly contributes to the high binding affinity to the DOPC membranes (0.53 µM) (Fig. 2A), since dissociation rates of both peptides are comparable, indicating the target role of lipid II (Fig. 2D).

Although we have to consider critically that the QCM measurements are based on a fixed peptide/lipid ratio and on an asymmetric supported model membrane of a covalently fixed first monolayer, which might have an impact on lipid II membrane insertion and accessibility, the high association of gallidermin indicates a certain tendency for membrane insertion. Changes in membrane permeability by both peptides or the ability to form pores into DOPC membranes were excluded by cyclic voltammetry measurements (data not shown).

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Detecting different modes of gallidermin membrane binding

To further evaluate the mechanisms behind the high binding affinity of gallidermin to pure DOPC membranes and the minor effects of lipid II in increasing the binding, a SAW biosensor technique was applied. This technology is based on the changes of phase and amplitude of surface acoustic waves due to binding processes of e.g. peptides on the surface of a sensor chip, and further allows distinguishing signals from mass changes and viscoelastic properties. The number of SAW binding studies is limited and only one recent study focused on the application of SAW in the field of antimicrobial peptide binding to model membranes [START_REF] Andrä | Surface acoustic wave biosensor as a tool to study the interaction of antimicrobial peptides with phospholipid and lipopolysaccharide model membranes[END_REF].

The supported membranes were created by spreading of liposomes at the sensors. In

Peptide interaction with pure DOPC membranes detected by ITC

The present data indicate strong differences of gallidermin and vancomycin in the mode of membrane interaction, although both peptides share the targeting of lipid II as basis for antibiotic activity. To further evaluate the mode of membrane interaction of both peptides, ITC as promising thermoanalytical technique was applied here as already described for other peptide membrane interactions, such as penetratin [START_REF] Binder | Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes[END_REF] or magainin [START_REF] Wenk | Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation[END_REF]. A recently published ITC study of vancomycin binding to synthetic fragments of the cell wall peptidoglycans confirmed a 1:1 binding stochiometry between vancomycin and the D-Ala-D-Ala moiety [START_REF] Rekharsky | Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall[END_REF]. Breukink et al. investigated the influence of membrane charge on the binding of nisin by ITC [START_REF] Breukink | Binding of Nisin Z to bilayer vesicles as determined with isothermal titration calorimetry[END_REF]. Since no reference data exist on the membrane interactions of gallidermin, the experimental parameters were selected according to former ITC investigations of peptide-lipid interactions by Seelig et al. [START_REF] Seelig | Titration calorimetry of lipid-peptide interactions[END_REF]. Referring to these established experiments and the fact that the higher concentrated reactant has to be injected into the lower concentrated one, DOPC liposomes were titrated into the peptide solution.

At each titration step, 150 nmol DOPC liposomes were added to an initial amount of 143 nmol peptide. Every further injection increased the molar ratio of lipid:peptide by the value of approximately 1. Although this sequence of peptide membrane contacts is not in accordance to the conditions of the biosensor measurements before, the resulting ITC scan should represent a comprehensive image to interpret the type of interactions. With respect to the high concentrations of peptide used, crystallization effects could be excluded considering the sufficient solubility of the peptide in aqueous solution (2 mg/mL).

Gallidermin titration with DOPC vesicles displayed a typical sigmoidal ITC trace of exothermic peaks (Fig. 4A). The heat rate decreased during the titration until reaching constant values, indicating a saturation of peptide-membrane interaction. In contrast to gallidermin, almost no changes in the heat rate of vancomycin titration with DOPC vesicles were detectable indicating only minor interactions (Fig. 4B). This is also reflected in about tenfold lower enthalpic changes of vancomycin compared to gallidermin (-508 ±

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These differences reflect the biosensor binding studies and refer that the exothermic peaks represent peptide membrane interaction. The trace of titration peaks of gallidermin allow for the calculation of a binding affinity of 3.28 µM, which is lower, but in a comparable range of the QCM data (0.53 µM). The differences might be explained by the experimental parameters, such as peptide/lipid ratio, or planar vs. liposomal model membranes.

To exclude that these findings were related to pH effects, the ITC measurements were performed under indicated buffer conditions (pH 7.4). The results confirmed that gallidermin displayed a pronounced interaction with the model membranes, while vancomycin in turn showed no membrane interaction; not even at higher temperatures.

To further focus on the mode of peptide membrane interaction, a fluorescence assay on the basis of merocyanine 540 was applied. MC 540 interacts with membranes changing its spectral characteristics according to the membrane fluidity. An increase in membrane fluidity corresponds with higher fluorescence emission at 584 nm [START_REF] Williamson | Merocyanine 540, a fluorescent probe sensitive to lipid packing[END_REF]. Peptide insertion into the liquid expanded DOPC bilayer should induce a condensing effect on the membrane order. Consequently, a resulting rigidification of the liposomes should be followed by a fluorescence decrease.

In accordance to the ITC experiments, gallidermin was added to MC540-pretreated DOPC liposomes at a molar ratio of 1:10 (representing the tenth titration peak), which induced a fluorescence decrease by 43% (reduction from 282 to 160 fluorescence units). In contrast, similar amounts of vancomycin had no influence on the fluorescence (248 to 259 u, corresponding to an increase of 4 %) . Further increase of gallidermin concentration to equimolar ratio reduced the emission by 55% (128 u), while vancomycin has only a minor effect at this concentration (187 u). These data strongly support the assumption derived from the SAW amplitude measurements (Fig. 3B, upper line) that gallidermin inserts into the membrane causing a rigidification, which is not true for vancomycin.

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ITC investigations on peptide interaction with DOPC/lipid II membranes

To investigate, whether the targeting of lipid II has an impact on the ITC traces and thus to obtain more interpretation of the scans in Figure 4, experiments were continued titrating gallidermin and vancomycin with DOPC liposomes containing 0.1 mol% lipid II (Fig. 5).

The exothermic trace of gallidermin (Fig. 5A) displayed a slight change in the curve shape compared to pure DOPC (Fig. 4A) suggesting that a saturation of the reaction occurred at lower concentrations. The changes in the heat rate were slightly diminished although the blank values were higher. Similar data could be obtained considering the influence of buffer or changed temperature in the ITC experiments (not shown). Lipid II has a certain impact on gallidermin binding, but it is not evident whether these changes represent direct lipid II binding of gallidermin, or membrane changes induced by lipid II.

Higher blank levels were also evident in the vancomycin titration trace (Fig. 5B). The change in enthalpy data (gallidermin -5455 cal/mol vs. -3018 cal/mol; vancomycin -508 cal/mol vs. -1353 cal/mol) are also not indicative for lipid II recognition of both peptides.

However, this does not exclude an interaction between vancomycin and lipid II, but demonstrate that in this case, the ITC method is not sensitive enough to detected the targetmediated binding of vancomycin and gallidermin. Furthermore, this finding is no contradiction to the data of Rekharsky et al. [START_REF] Rekharsky | Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall[END_REF] who detected the interaction of vancomycin with much higher concentrations of D-Ala-D-Ala, compared to lipid II concentration in model membranes in our study.

To confirm that the minor changes in the ITC scans in presence of lipid II were induced rather by indirect effects of the lipid II isoprenoid chain than by target recognition of the lipid II head group, we incorporated the lipid II membrane anchor C55P into the DOPC liposomes. Since the peptide side chain, the sugar moiety, and the second phosphate are missing, a specific recognition by gallidermin or vancomycin can be excluded. Gallidermin

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displayed a binding affinity of 0.21 µM ± 0.07 µM to DOPC/C55P membranes in QCM experiments. This is comparable to gallidermin affinity to both, pure and lipid II containing DOPC membranes, which indicates no specific effects of C55P.

The gallidermin trace in presence of C55P (Figure 6) is comparable to the lipid II scan, the higher exothermic peaks (titration steps 10-18) imply a slightly stronger interaction, most likely incorporation of gallidermin into the membrane than in presence of lipid II. The ITC trace of vancomycin is identical to pure DOPC indicating no membrane interaction.

Therefore, these data suggest that the curve characteristics in Figure 5 are mainly induced by membrane effects in the hydrophobic moiety. Recognition of lipid II by both peptides can not be detected by ITC.

To further differentiate the effects of lipid II and C55P, we calculated the molar amount of membrane interacting gallidermin X p for each injection k according to Eq. 1, which is described in detail in [START_REF] Wenk | Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation[END_REF][START_REF] Seelig | Titration calorimetry of lipid-peptide interactions[END_REF].

n k k k p k p k p h h n n X 1 1 ) ( ) ( / ( 1
)
Plotting the molar amount of membrane interacting gallidermin X p against the first 25 titration steps (Fig. 7) illustrates that lipid II and C55P reduce the gallidermin interaction with DOPC membranes to a similar extent. This suggests to a dominant effect of the isoprenoid chain of lipid II and C55P on the membrane and thus, on gallidermin membrane insertion. This finding totally reflects the SAW binding data that gallidermin membrane insertion occurs at higher concentrations and to a lower degree, when lipid II is present.

Taking the slope from step 10 to 20 as an indicator for membrane interaction, (0.0343; 0.0285; and 0.0181 for DOPC, DOPC/C55P, DOPC/lipid II, respectively), the calculated affinities from the titration traces of gallidermin are in good agreement (3.28 µM; 7.09 µM; and 18.9 µM). The slightly lower slope in the presence of lipid II compared to C55P can reflect the recognition of lipid II interfering with unspecific membrane internalization.

These data also allow for the interpretation that membrane inserted gallidermin disturb membrane integrity. In addition to the "classical" targeting of lipid II at the membrane surface, these inner membrane effects might also add to the sequestration of lipid II from the sites of cell wall biosynthesis and thus contribute to the antibiotic effects of gallidermin [START_REF] Hasper | An alternative bactericidal mechanism of action for lantibiotic peptides that target lipid II[END_REF].

Conclusions

Overall, the data presented here provide evidence for non-targeted membrane activities of gallidermin in synergy with lipid II mediated interactions leading to cell wall inhibition in combination with membrane disturbing effects. Both activities will be affected by the bacterial membrane characteristics, which might therefore be key for the activity against individual strains. In the case of vancomycin that does not interact with the membrane, other factors e.g. accessibility of lipid II in the bacterial membrane are the main determinants of antimicrobial activity. The model membrane approaches used here are powerful tools to analyze membrane activities of antimicrobial peptides. Especially the SAW technique is a sensitive method to detect peptide-target interactions. The presence of lipid II possesses only marginal effects, the titration traces do not allow to derive targeting effects of both peptides to lipid II.

Legends to Figures

  applied with the two frequency measurement mode between 144 MHz and 152 MHz. The gold surface of the SiO 2 sensor chips was functionalized by self-assembled monolayers of 11-amino-1-undecanethiol hydrochloride (2 mM) overnight. After washing with ethanol and Millipore ® water, the sensor chips were placed into the chip reader unit of the SAW device and the frequency optimum was determined. The measurement temperature was 25°C and the flow rate was set to 40 µL/min. After an equilibration time of 10 min, 300 µL of liposome solution (1 mM) were added. Subsequent peptide injections of 150 µL were performed in a concentration range from 10 -13 M to 10 -6 M. Changes in phase shift and amplitude were registered online.All measurements were carried out with a Microcal VP-ITC calorimeter (Microcal, Northampton, USA). The VP-ITC device was electrically calibrated. All samples were degassed and temperated before measurement. 25°C was chosen as the experimental temperature. The reference power was set to 8 µcal/sec, using a syringe stirring speed of 350 rpm. Titrations were performed by a number of 50 injections of each 5 µL steps of LUV aliquots (30 mM) into the sample cell containing 1.43 mL peptide at a concentration of 100 μM. The change in heat rate during the titration steps was registered in real time.

  contrast to the fixed peptide/lipid ratio in the QCM experiments, step like increasing concentrations of gallidermin were allowed to interact with the membrane, starting with 0.1 pM. The phase changes, indicating the interaction of gallidermin with pure DOPC (Fig.3A, upper line) displayed two minor signals at 0.1 pM and 1 pM followed by a very strong phase shift at 10 pM concentration (dashed line). The associated changes in amplitude (Figure3B, upper line) indicate the viscoelasticity of the surface bound structures. The increase of amplitude at 10 pM gallidermin (dashed line) can clearly be addressed to a membrane rigidification and suggests a gallidermin insertion into the membrane. This supports the assumption that gallidermin tends to insert into the membrane, which occurs mainly unique at a certain threshold concentration, obviously leading to a membrane saturation. Vancomycin displayed no interaction under identical conditions (not shown).Considering the lipid II containing membrane, gallidermin revealed a different mode of interaction compared to the pure DOPC. Small signals in phase shift (Fig.3A, lower line) indicate weak binding events at 1 pM and 10 pM and were followed by a stronger and reversible signal at 100 pM gallidermin. These signals indicate the binding of gallidermin to lipid II. To further evaluate these findings, the most prominent phase shift at 100 pMA C C E P T E D M A N U S C R I P TACCEPTED MANUSCRIPT gallidermin (dotted line) was analyzed with respect to the peptide/lipid II ratio. The number of lipid II molecules (0.1 mol% in the lipid matrix) at the sensor surface was calculated, dividing the sensitive surface by the mean phospholipid head group size, to be about 1.4×10 10 lipid II molecules. This excellently fits with the peak at 100 pM gallidermin, which corresponds to about 1.1×10 10 gallidermin molecules in the measurement compartment. Thus, the lipid II recognition of gallidermin is confirmed and occured at a binding ratio of roughly 1 : 1. Further insight is given by the course of amplitude of this measurement (Fig.3B, lower line). The binding of gallidermin to lipid II at the three lowest concentrations (0.1 -10 pM) is associated with small drops in amplitude, indicating an increase in viscoelasticity. This is also true for the dominat binding at 100 pM (dotted line), but after an initial drop, the amplitude increases evidently.This refers to the fact that lipid II binding of gallidermin is superimposed by membrane insertion of the peptide. Interestingly, the concentration range of the gallidermin lipid II binding signals in the SAW measurements (0.1 nM gallidermin) is in the range of the in vivo activity of gallidermin against sensitive strains, determined as MICs; i.e. Lactobacillus lactis HP and Lactobacillus fermentum ATCC 9338 , 5 nM and 1 nM, respectively. Summarizing, the SAW measurements give clear indications for a lipid II recognition of gallidermin and dominant binding at roughly equimolar peptide/lipid II ratio and simultaneous membrane insertion of gallidermin. The reduction of gallidermin membrane insertion in presence of lipid II is obviously caused by membrane stabilizing effects of lipid II, which we have recently illustrated microscopically by reduced lipid lateral diffusion rates in presence of lipid II [25].

Figure 1 :

 1 Figure 1: Structure of the bacterial cell wall precursor lipid II and it target moieties for the

Figure 2 :

 2 Figure 2: Detection of the kinetic constants of vancomycin and gallidermin binding to model

Figure 3 :

 3 Figure 3: SAW experiments of gallidermin binding to pure DOPC and DOPC containing 0.1 mol%

Figure 4 :

 4 Figure 4: Representative ITC-scans of titrating DOPC liposomes into gallidermin (A) or

Figure 5 :

 5 Figure 5: ITC traces of titrating DOPC/0.1 mol% lipid II into gallidermin (A) and vancomycin (B).

Figure 6 :

 6 Figure 6: ITC traces of titrating DOPC/0.1 mol% C55P into gallidermin (A) and vancomycin (B) to

Figure 7 :

 7 Figure 7: Comparison of gallidermin titration with DOPC, DOPC/0.1 mol% lipid II or

Fig. 1 A

 1 Fig. 1

  Fig. 3

Fig. 4 A

 4 Fig. 4

Fig. 5 A

 5 Fig. 5

Fig. 6 A

 6 Fig. 6

Fig. 7 A

 7 Fig. 7

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft, BE2242/2-1, FOR 854, and GRK 677. The authors are thankful to Prof. Joachim Seelig for helpful discussions concerning the ITC data, Dr. Thomas Gronewold for help in interpretation of the SAW data, and Prof. Hans-Georg Sahl for providing the lipid II. We thank H.

Falkenstein-Paul for excellent technical assistance, and M. Josten for mass determination of the peptides.

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B Vileno
email: vileno@unistra.fr

S Jeney

A Sienkiewicz

P R Marcoux

L M Miller

L Forró

Evidence of lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated by fullerols

Keywords: IR, Infrared Spectroscopic Microscopy, Synchrotron, Fullerols, Reactive Oxygen Species, Oxidative Stress

An Oxidative Stress (OS) state is characterized by the generation of Reactive Oxygen Species (ROS) in a biological system above its capacity to counterbalance them [1]. Exposure to OS induces the accumulation of intracellular ROS, which in turn causes cell damage in the form of protein, lipid, and/or DNA oxidations. Such conditions are believed to be linked to numerous diseases or simply to the ageing of tissues. However, the controlled generation of ROS via photosensitizing drugs or photosensitizers (PS) is now widely used to treat various tumors and other infections [2,3]. Here we present a method to track the chemical changes in a cell after

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particular, elevated ROS levels in cells are associated with the aging process [4], as well as with many pathological disease processes [1,5]. Among various ROS involved in bio-oxidative and bio-photo-oxidative processes, 1 g plays a significant role. In particular, 1 g is involved in lightinduced photo-damage to the eyes and skin [6]. 1 g is known to be almost as reactive as atomic oxygen and to react directly with proteins and unsaturated lipids located in the cytosol and cell membranes to give the corresponding peroxides. Consequently, cell apoptosis, bacterial and viral inactivation are identified effects of 1 g action [7,8] with higher yields than hydroxide (OH • ) or superoxide (O 2

•─

) radicals [9].

Pristine fullerene (C 60 ), due to its unique electronic -system, can generate 1 g and other ROS in organic solvents under UV or visible light illumination [10]. It has also been shown that the poly-hydroxylated and highly water-soluble derivatives of C 60 , also known as fullerols, are potent photosensitizers of ROS. In particular, evidence of generation of superoxide radicals via the so-called Type I mechanism, as well as 1 g via an effective resonant energy transfer (Type I photosensitization pathway) was demonstrated for fullerol C 60 (OH) 24 [11]. In a previous studywe have shown that a custom-synthesized fullerol (C 60 (OH) 19 (ONa) 17 ,18H 2 O) can efficiently generate 1 g in aqueous milieus under visible light illumination and also pointed to a strong photo-toxicity of this compound towards cells [12] and cell compounds [13].

In this work, the oxidative stress on living monkey fibroblasts was mediated via ROS, which was photo-sensitized in the presence of the above-mentioned fullerol C 60 (OH) 19 (ONa) 17 ,18H 2 O [12].

First, the generation of 1 g by this water-soluble derivative of C 60 via Type II photodynamic process was confirmed in a cell-free EPR experiment using a 1 g selective substrate, 2,2,6,6-

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tetramethyl-4-piperidinol (TMP-OH). Then, the OS-induced biochemical alterations in living fibroblasts were tracked by Fourier Transform InfraRed Microspectroscopy (FTIRM) at the single cell level. To achieve the required subcellular spatial resolution, an IR beam size smaller than the cell size itself is needed. However, existing IRMS instruments using a conventional thermal (globar) source, encounter a Signal-to-Noise ratio (S/N) limitation when apertures confine the IR to an area of below 30 m 2 [14]. Hence, for single cell imaging, the InfraRed (IR) synchrotron source delivers the entire infrared wavelength at much higher brightness than a globar source [15]. For a 10 by 10 m 2 aperture, the S/N ratio is improved by a few orders of magnitude compared to globar sources [16]. In order to account for heterogeneities in living matter, we also looked for spectral anomalies for a given functional group in a population of at least 75 cells, allowing statistical sampling.

MATERIALS AND METHODS:

Fullerol synthesis and ROS generation. The fullerol synthesis was adapted from Chiang et al.

[17], based on the oxidation of C 60 under strongly acidic conditions. The synthesis and characterization is detailed in [12]. windows (13 mm diameter, 1 mm thickness, Crystran, UK) using an identical number of passages. CaF 2 is known to be non toxic to cells and fully transparent in the relevant IR range between 1000 and 4000 cm -1 . A density of 25000-30000 cells per window was deposited, and used for experiment before they reached confluent state (day 2-3), thus single cells were still distinguishable [21].

Protocol of Cell Irradiations. The cell-covered CaF 2 -windows were removed from the culture medium, rinsed in PBS, placed in the photosensitizing solution (i.e. PBS supplemented by 500 M C 60 (OH) 19 (ONa) 17 ), and then illuminated for three different exposure times (10, 20 and 30 min). Special care was taken not to heat the sample by the light source (Halogen Philips, 150W).

After exposure, the photosensitizing solution was removed, the cells were rinsed in PBS, incubated for 30 min in DMEM, rinsed again with PBS and left to dry at 4°C under a soft air flow. Once fully dried, the windows were carefully re-washed with distilled water in order to

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remove PBS salt crystals on the window surface, fully dried again and kept at 4°C until SIRMS experiments [22,23,24].

A Trypan Blue (TB, Sigma Aldrich) assay was used to probe the viability of the cells upon OS.

TB is negatively charged and typically does not penetrate a cell unless its membrane is damaged.

Consequently, all cells including this dye and thus having a blue stained cytoplasm are dead.

Inversely all viable cells exclude the dye and have a clear cytoplasm. Noteworthy, the TB assay cannot differentiate between a necrotic or apoptotic cell.

Synchrotron Infrared MicroSpectroscopy. Synchrotron-based Fourier Transform Infrared

Microspectroscopy (S-FTIRM) measurements were performed on dried cells prepared as described above. The S-FTIRM set-up [14] consists of a Thermo Nicolet Continum IR microscope coupled to a Thermo Nicolet Magna 860 FTIR (Thermo Nicolet Instrument, Madison, WI). An automated X-Y mapping stage allowed sample scanning with a step accuracy of 1 m. The IR beam, with an aperture of 7 x 7 to 10 x 10 m 2 , was positioned on the center of a single flat cell chosen by conventional optical microscopy, illuminating most of the cell.

Spectra were collected in transmission mode, at a spectral resolution of 4 cm -1 , and 128 to 256 scans were co-added to enhance the S/N ratio. Prior to each experiment, a background spectrum was taken at a clean cell-free place on the CaF 2 window and subtracted from the one with the sample. Data were analyzed using Omnic 7.4 (Thermo Fisher Scientific Inc), and Transform (Fortner Software). Few bad pixels were removed and replaced either by the mean value of their neighbor's pixel (e.g. for odd absorbance values) or set to zero (for negative values). It is worth noting that despite the high brightness of the IR source, the total energy delivered to a sample for

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an aperture of 10 by 10 m 2 is less than 5 mW [25]. The local rise of temperature under IR synchrotron irradiation has been determined to be lower than one degree [26]. Therefore, we assumed that heating had no effect on our experiment. Compared to the non-exposed fibroblast, the most striking change is the appearance of a peak centered at 1740 cm -1 (blue arrow). Nevertheless, the absolute increase of this peak could be due to a high number of lipid molecules and/or high lipid density in the cell. This could be especially the case when the aliphatic chains band (region 2995-2800 cm -1 , green arrow), which is connected to lipids, also increased. Therefore, we calculated the ratio between the C=O representative band and the lipids for N > 75 cells at three different time points T = 10, 20 and 30 min. Results are shown in Figure 3a. All values were normalized to control (1) where the cells were not subjected to any treatment. Control (2) and (3) were performed respectively in the presence of PS in the dark and after 20 min of illumination but without PS. Even though the distribution of the obtained values is relatively high, due to a strong heterogeneity among cells, the ratio between carbonyl ester groups in non-hydrogen bonded state and lipids, in each experimental group, were consistent and almost doubled after a 20 min OS exposure.

RESULTS

Kinetics

Specific protein-phosphorylation events have been previously demonstrated to occur during apoptosis and play an important role in the regulation of the programmed cell death mechanism

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ACCEPTED MANUSCRIPT [34]. Hence, we also calculated the ratio between the asymmetric phosphate stretching region, 1280-1180 cm -1 , and the Amide I peak at 1710-1600 cm -1 for N > 75 cells at three different time points T = 10, 20 and 30 min (Figure 3b).

To determine whether the differences in the means of the presented histograms are significant, we performed a two tails t-test comparing the control (1) with the 10, 20 and 30 min oxidative stress statistics. The data were expressed as means and standard errors. The obtained t values for for p = 0.1 or less were considered as significant. All our data fulfilled the imposed t-test conditions.

Single cell mapping. In order to localize characteristic spectral features at higher resolution we recorded an IR map of a single cell, by scanning the sample. The step size was 8 m and the aperture was set to 7 m, the diffraction-limited spatial resolution allowed a detailed mapping of a single cell with a size between ten and a few tens of a micrometer. Flat, well-spread cells were chosen by optical microscopy ensuring that the observed variations in intensity arose mostly from local concentration changes rather than from local thickness changes. Different distributions of the chemical functional groups are presented in Figure 4

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cm -1 area. The protein distribution (C) for both, treated and non-treated cells exhibited a relatively homogenous distribution and high intensity at the cell center. This corresponded to the thickest part of the cell where the nucleus was also present, and in which a high protein density is found [35]. In contrast, the lipid distribution (B) was more spread, confirming that lipids are well distributed among the intracellular compartments, like the endoplasmic reticulum, the Golgi apparatus, the nuclear and cell membrane. No significant changes in either distribution were visible between the control and the treated cell, except that the treated cell was thicker and gave a stronger signal.

In order to normalize such thickness differences in cells, and detect relative changes in lipid peroxidation and protein phosphorylation from a non-treated to a treated cell (panels D and E in Figure 4), we also represented the peak ratios: 1730-1760 cm -1 (C=O) over 2995-2800 cm -1 (lipids) and 1280-1180 cm -1 (PO 2 -) over 1710-1600 cm -1 (Amide I). Both signatures were significantly enhanced upon ROS treatment (see Figure 5 panel D and E respectively), as it was already observed in single cell spectra (Figure 2, bottom).

DISCUSSION AND CONCLUSION

We have shown that 1 g is generated within the timeframe of fullerol illumination (Figure 1b).

This allowed us to conclude that the detected spectrum anomalies arising in the cells are largely due to the effect of 1 g . The most significant spectrum anomaly observed in cells exposed to OS was an increase of the peak around 1740 cm -1 , after 20 min of exposure to OS (Figure 3a). This

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peak increase was referred to an IR fingerprint of necrotic cells [16,35]. In our case, it can be explained by the generation and accumulation of lipid peroxidation end products, known as Reactive Carbonyl Compound (RCC) such as e.g. malondialdehyde (MDA) [37] or acrolein [38].

Moreover, according to the chemical map of the cell in Figure 4 and 5 panel D, such increases are predominantly located in the cell periphery, distant from the nucleus (highlighted by a white dashed circle). Hence this pattern can be ascribed to the increase of oxidized lipids present in the membrane of dying cells. This feature, localized around the nucleus, is in excellent agreement with the study performed on necrotic cells by Dumas et al. [16].

On the other hand, the ratio between the peak partially associated with phosphorylated proteins (asymmetric phosphate stretching band, 1280-1180cm -1 ) and the protein peak (Amide I, 1710-1600 cm -1 ) integrated intensities started to increase earlier: after only 10 min of exposure to OS (as shown in Figure 3b). Specific protein-phosphorylation events are known to occur during apoptosis and also play an important role in the regulation of programmed cell death [39,40].

Cell mapping in Panels E (Figure 4 and 5) shows an increase in the phosphate contribution, spread over the whole cell, possibly indicating that cytoskeletal proteins were targeted during this apoptotic process. In summary, exposure to oxidative stress, and in particular 1 g , induces a cascade of lipid peroxidation and phosphorylation reactions within the observed cells. First, protein phosphorylation occurs within the cytoplasm and nucleus and later lipid phosphorylation of membrane structures. However, attributing this OS-induced phosphorylation to an apoptotic or necrotic reaction remains still open. It is very likely that a combination of both pathways exists.

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Concerning the distribution in the value obtained in our statistical sampling, it is worth noting that the cells studied after exposure to oxidative stress, might have been those which better resisted the photo-mediated action. Considering the experimental procedure, those cells with more damage had a higher likelihood to detach from the surface and were then washed away during the multiple rinsing steps. Therefore, they were not considered in this study, which compromised the statistics. In conclusion, we successfully used FTIRM to detect small OSmediated biochemical changes in cells. Especially, lipid peroxidation and protein phosphorylation have been identified as OS fingerprint. These features were characterized by an increase of the ester (1740 cm -1 ) and phosphate asymmetric stretching (1240 cm -1 ) related peaks respectively.

A C C E P T E D M A N U S C R I P T between the phosphorylated protein peak area (asymmetric phosphate stretching band, 1280-1180 cm -1 ) and the protein peak area (Amide I, 1710-1600 cm -1 ). Controls were the following:

(

Figure 1b .

 1b Figure 1b. As shown in the inset, the observed EPR spectra are 1:1:1 triplets, indicative of TEMPOL, the paramagnetic product of the 1 g attack on TMP-OH. The amplitude of the

Figure 2 (

 2 Figure 2 (bottom spectrum) shows a typical IR spectrum of a single fibroblast after 30 min of

  and 5, for a non-treated fibroblast and for a fibroblast cell after a maximal exposure to photo-mediated OS (30 min / 500 M PS in oxygenated PBS solution): (A) optical image of the cell, (B) distribution map of lipids computed from the integration of the aliphatic C-H stretching band area (2995-2800 cm -1 ), (C) distribution map of proteins represented by the integration of the Amide I band area (1710-1600cm -1 ), (D) ester distribution mapped by integration of the carbonyl ester group stretching band (1730-1760 cm -1 ) and (E) asymmetric phosphate stretching represented by the 1280-1180 A C C E P T E D M A N U S C R I P T

  was already observed by Holman et al. in dying fibroblasts, and attributed to the non-hydrogenbonded ester carbonyl stretching mode in phospholipids [36]. Moreover, in previous studies this A C C E P T E D M A N U S C R I P T

Figure 1 :

 1 FIGURE CAPTIONS

Figure 2 :

 2 Figure 2: Infrared spectra of dried single monkey fibroblasts for: (top) a non-treated cell (ctrl)

Figure 3 :

 3 Figure 3: Histograms of the integrated area ratio between (a) the ester carbonyl stretching mode (1760-1730 cm -1 region) and the aliphatic C-H stretch region (2995-2800 cm -1 region) and (b)

Figure 4 :

 4 Figure 4: Single cell mapping of the functional group distribution for a non-treated (CTRL)

Figure 5 :

 5 Figure 5: Single cell mapping of the functional group distribution for a non-treated (CTRL)

of ROS-generation by C 60 (OH) 19 (ONa) 17 .

		The EPR measurements confirmed the
	photosensitization of 1	g in PBS buffered H 2 O solutions in the presence of fullerol
	C 60 (OH) 19 (ONa) 17 ,18 H 2 O. The typical EPR results on reactive scavenging of 1	g are shown in

ACKNOWLEDGEMENTS

The authors are also particularly thankful to Randy Smith for his technical assistance and Dr. Titusz Feher for his constructive discussions. The NSLS is supported by the United States Department of Energy under contract DE-AC02-98CH10886. These studies were also partially supported by the Swiss National Science Foundation, project No. 205320 -112164, "Biomolecules under stress: ESR in vitro study" (A.S. and L.F.).

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