Yiannis E Tsekouras

Faidon Magkos

Konstantinos I Prentzas

Konstantinos N Basioukas

Stergoula G Matsama

Amalia E Yanni

Stavros A Kavouras

Ph.D Labros S Sidossis
email: lsidossis@hua.gr

A single

Keywords: lipid kinetics, hepatic metabolism, triglyceride, acute exercise

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Increased fasting plasma triacylglycerol (TAG) concentrations are associated with higher risk for coronary heart disease, independently of other major lipid risk factors [START_REF] Hokanson | Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a metaanalysis of population-based prospective studies[END_REF]. Postabsorptive hypertriacylglycerolemia typically results from an imbalance between the rate of very lowdensity lipoprotein (VLDL) TAG secretion from the liver and the rate of VLDL-TAG removal from the circulation [START_REF] Boberg | Splanchnic secretion rates of plasma triglycerides and total and splanchnic turnover of plasma free fatty acids in men with normo-and hypertriglyceridaemia[END_REF][START_REF] Reaven | Kinetics of triglyceride turnover of very low density lipoproteins of human plasma[END_REF]. It was demonstrated recently that a single bout of prolonged aerobic exercise (90-120 min at moderate intensity) decreases basal VLDL-TAG concentration the next day by increasing the rate of whole-body VLDL-TAG clearance [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Tsekouras | A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men[END_REF]. This effect is probably mediated by the secretion of fewer but TAG-richer VLDL particles after exercise [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF], which are hydrolyzed faster by lipoprotein lipase (LPL) in vitro [START_REF] Fisher | Human triacylglycerol-rich lipoprotein subfractions as substrates for lipoprotein lipase[END_REF] and removed faster from the circulation in vivo [START_REF] Streja | Triglyceride removal from very low density lipoproteins in vivo as a function of their triglyceride content[END_REF]. In addition, possible transient increases in LPL activity in skeletal muscle (but not adipose tissue) after exercise [START_REF] Seip | Skeletal muscle lipoprotein lipase: molecular regulation and physiological effects in relation to exercise[END_REF] may further augment TAG clearance [START_REF] Kiens | Lipoprotein metabolism influenced by training-induced changes in human skeletal muscle[END_REF]10].

Although it is well established that single bouts of prolonged (≥ 90 min), moderate-intensity aerobic exercise significantly reduce basal plasma TAG concentrations the following day in a manner that depends on the total energy expenditure of exercise [11][12][13][14], there is currently no consensus regarding the effects of resistance exercise. Petitt et al. [15] showed that a single resistance exercise bout (total energy expenditure ~1.7 MJ) lowered fasting plasma TAG concentrations the next day in recreationally weight-trained men and women, and the effect was actually greater than that of an aerobic exercise bout of similar energy expenditure. However, others have failed to observe a TAG-lowering effect in the fasting state after single bouts of resistance exercise ranging in total energy expenditure from ~0.6 MJ to 2.6 MJ [START_REF] Burns | A single session of resistance exercise does not reduce postprandial lipaemia[END_REF][START_REF] Shannon | Resistance exercise and postprandial lipemia: The dose effect of differing volumes of acute resistance exercise bouts[END_REF][START_REF] Zafeiridis | Effects of low-and high-volume resistance exercise on postprandial lipaemia[END_REF], and Burns et al. [19] found increased postprandial plasma TAG concentrations immediately after resistance exercise, though the effect was transient and disappeared ~8 hours following meal ingestion.

The effect of a single bout of resistance exercise on fasting plasma TAG concentration is therefore not clear. Furthermore, all previous studies examined TAG metabolism in response to high-fat meals [15][START_REF] Burns | A single session of resistance exercise does not reduce postprandial lipaemia[END_REF][START_REF] Shannon | Resistance exercise and postprandial lipemia: The dose effect of differing volumes of acute resistance exercise bouts[END_REF][START_REF] Zafeiridis | Effects of low-and high-volume resistance exercise on postprandial lipaemia[END_REF][19]; there is currently no information regarding the effects of resistance minutes. The test was terminated at 80% of heart rate reserve, and VO 2 peak was predicted from the oxygen consumption / heart rate relationship [21]. All volunteers were familiarized with the isokinetic resistance exercise protocol on a separate day, and three maximum intensity repetitions were performed to determine peak torque production for each exercise.

Experimental protocol

Volunteers performed two time-matched trials (rest and exercise) approximately one week apart, in a randomized counterbalanced design. For the exercise trial, they were admitted to the laboratory on the evening before the isotope infusion study and performed a single bout of resistance exercise on an isokinetic dynamometer (BIODEX System 3 Pro, Shirley, NY). The exercise protocol was designed so as to involve whole-body resistance exercise, consisting of 3 sets of 10 repetitions at 80% of maximum peak torque production, for a total of 12 exercises (Table 1). All participants had visible contact with a bar chart on the monitor that indicated performance for each repetition and obtained verbal feedback to achieve the target. The exercise protocol was performed for each limb separately and the resting period between exercises was 2 minutes. The order in which the individual exercises were performed was the same for all participants. Total exercise duration was 90 ± 1 min. Heart rate was monitored continuously by a telemetric heart rate monitor (Polar Accurex Plus, Finland). Energy expenditure was measured by indirect calorimetry using a face mask throughout exercise, as done in previous studies examining the effect of resistance exercise on TAG metabolism [15,[START_REF] Zafeiridis | Effects of low-and high-volume resistance exercise on postprandial lipaemia[END_REF]. Despite that the short and intermittent duration of resistance exercise invalidates the typical assumptions of indirect calorimetry, since the respiratory quotient (RQ) is consistently ≥ 1, using a fixed caloric cost for the volume of O 2 consumed during exercise, as done by other investigators [START_REF] Burns | A single session of resistance exercise does not reduce postprandial lipaemia[END_REF]19], yielded slightly higher (by ≤15%) estimations for the total energy expenditure of our exercise session;

Diet assessment and control

Upon entry into the study, volunteers received instructions on how to record food and beverage intake and provided a detailed recording of all nutrient intake for the 3 days before the first trial, including the last meal (dinner) consumed after the completion of the exercise or resting session (whichever came first). They were then instructed to reproduce the same diet for the 3 days preceding the second trial. None of the subjects reported any deviation from the dietary plan.

Food journals were analyzed by using nutrition analysis software (Nutritionist Five, FirstDataBank, San Bruno, CA). No alcohol or caffeine intake was permitted for two days before each isotope infusion study. Also, no caloric consumption was permitted during the exercise session or the equivalent period of rest. Dinner was completed within 2 h after cessation of the

VLDL preparation and analyses

The VLDL fraction was prepared as previously described [START_REF] Tsekouras | A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men[END_REF]. Briefly, ~2 ml of plasma was

Isolation of TAG in VLDL particles

Aliquots (~750 μl) of the isolated VLDL fractions were deproteinized with 3 ml acetone, centrifuged (3000 rpm for 15 min at 4°C), and the supernatant was dried under vacuum monoexponential approach, after logarithmically transforming the raw TTR data [START_REF] Lemieux | A stable isotope method using a [ 2 H 5 ]glycerol bolus to measure very low density lipoprotein triglyceride kinetics in humans[END_REF]. The total rate of hepatic VLDL-TAG secretion was calculated as: total VLDL-TAG secretion (μmol/min) = FCR × C × PV / 60, where C is the concentration of VLDL-TAG in plasma (μmol/ml) and PV is the plasma volume (ml), which was calculated as 55 ml per kg of fat-free mass [START_REF] Boer | Estimated lean body mass as an index for normalization of body fluid volumes in humans[END_REF] 1.

Metabolic rate and substrate oxidation during fasting

Basal VO 2 (exercise: 262 ± 10 ml/min and rest: 252 ± 10 ml/min, P = 0.25) and hence RMR (exercise: 1764 ± 80 kcal/day and rest: 1706 ± 65 kcal/day, P = 0.16) were not significantly different the morning after the two interventions. Likewise, there was no effect of evening exercise compared with rest on basal RQ (0.75 ± 0.01 and 0.77 ± 0.02, respectively, P = 0.32) and whole-body fat oxidation rate (0.093 ± 0.012 and 0.078 ± 0.009 g/min, respectively, P = 0.12).

Substrate and insulin concentrations during fasting

Plasma volume changes between the two trials were minimal (2 ± 1%, P = 0.14). Evening resistance exercise, compared with rest, did not affect serum insulin and plasma glucose concentrations and therefore whole-body insulin sensitivity (HOMA-IR index), nor plasma total, HDL-, and LDL-cholesterol and serum lactate dehydrogenase concentrations (Table 2). Plasma FFA, serum beta-hydroxybutyrate and creatine kinase concentrations were significantly higher the morning after exercise than rest, whereas total plasma TAG, VLDL-TAG and VLDL-apoB- 2).

Basal VLDL-TAG kinetics

In both trials, plasma VLDL-TAG concentrations remained constant throughout the 6-h sampling period (Figure 1), indicating a metabolic steady state. The time-courses of glycerol TTR in VLDL-TAG and logarithmically transformed TTR data are shown in Figure 2. The FCR of VLDL-TAG was significantly higher after exercise than rest (0.56 ± 0.05 and 0.46 ± 0.05 pools/h, respectively, P = 0.007). Exercise did not affect the rate of hepatic secretion of VLDL-TAG (P = 0.31), but increased VLDL-TAG plasma clearance rate by 26 ± 8% (P = 0.004) compared with rest (Figure 3). The mean residence time of VLDL-TAG in the circulation was significantly shorter after exercise than rest (113 ± 10 and 144 ± 18 min, respectively, P = 0.02).

DISCUSSION

In the present study, we examined basal VLDL-TAG kinetics the morning after a single, evening bout of strenuous resistance exercise or an equivalent period of rest in healthy, untrained young men. We found that resistance exercise lowered fasting plasma VLDL-TAG concentrations by ~30% compared with rest, due to enhanced VLDL-TAG removal from the circulation (by ~25%), resulting in ~30 min shorter residence time of VLDL-TAG in the bloodstream. This effect was independent of changes in glucose and insulin concentrations and insulin sensitivity (i.e., HOMA-IR index). Resistance exercise did not affect the rate of VLDL-TAG secretion from the liver, even though is greatly augmented FFA availability (as indicated by the higher FFA concentrations), likely due to increased hepatic fatty acid oxidation (as indicated by the higher beta-hydroxybutyrate concentrations) and perhaps also use of FFA for tissue TAG repletion.

These results suggest that the hypotriacylglycerolemic effect of acute resistance exercise is qualitatively and quantitatively similar to that reported previously for endurance exercise [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Tsekouras | A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men[END_REF].

Single bouts of prolonged (≥ 90 min), moderate-intensity aerobic exercise lower fasting plasma TAG concentrations the following day, provided that sufficient energy be expended during 11 for coronary heart disease [START_REF] Hurley | Effects of weight training on risk factors for coronary artery disease[END_REF], but the effects of acute resistance exercise on TAG metabolism are not well understood. Results from previous studies using a similar protocol to ours are inconsistent: a single bout of resistance exercise of approximately the same duration (~90 min) and total energy expenditure (1.7-2.3 MJ) has been reported to either decrease [15] or not affect [START_REF] Burns | A single session of resistance exercise does not reduce postprandial lipaemia[END_REF] fasting and postprandial plasma TAG concentrations the next morning. Others reported unchanged fasting but reduced postprandial TAG concentrations, when measurements were made ≥ 14 h after the cessation of resistance exercise sessions of even shorter duration (40-50 min) and total energy expenditure (0.75-1.4 MJ) [START_REF] Zafeiridis | Effects of low-and high-volume resistance exercise on postprandial lipaemia[END_REF], whereas an increase in postprandial plasma TAG concentrations was observed when measurements were made immediately postexercise [19].

The reasons for these inconsistent results are not clear, nor can they be inferred from our study.

One possible explanation may be differences in energy balance between the rest and exercise trials. It was recently shown that the hypotriacylglycerolemic effect of aerobic exercise in the fasting and postprandial states is abolished when the energy expended during exercise is replaced by overfeeding [START_REF] Burton | Energy replacement attenuates the effects of prior moderate exercise on postprandial metabolism in overweight/obese men[END_REF], and single bouts of resistance exercise varying in duration from 20 to 90 min and total energy expenditure from 0.6 to 2.6 MJ do not affect fasting and postprandial TAG concentrations in the face of increased caloric intake [START_REF] Shannon | Resistance exercise and postprandial lipemia: The dose effect of differing volumes of acute resistance exercise bouts[END_REF]. Hence the inherent inability to accurately quantify the energy expenditure of resistance exercise by means of indirect calorimetry [15,[START_REF] Zafeiridis | Effects of low-and high-volume resistance exercise on postprandial lipaemia[END_REF] or otherwise [START_REF] Burns | A single session of resistance exercise does not reduce postprandial lipaemia[END_REF][START_REF] Shannon | Resistance exercise and postprandial lipemia: The dose effect of differing volumes of acute resistance exercise bouts[END_REF]19] and possible differences in dietary energy intake during the days preceding each trial might have contributed to the reported inconsistent results.

Clearly, the role of energy balance and its components (exercise energy expenditure and dietary energy intake) in mediating the effects of exercise on TAG metabolism warrant further investigation. In the present study, controlling of the diet for the 3 days preceding the two tracer infusions (by recording all food and beverage intake before the first trial and replicating this diet before the second trial) ensured that there were no major differences in energy content and Our results indicate, for the first time in humans in vivo, that increased VLDL-TAG removal rate from the circulation mediates the TAG-lowering effect of resistance exercise. We cannot ascertain the site/tissue responsible for this effect, since VLDL-TAG clearance refers to all possible routes of VLDL-TAG removal from plasma. This includes hydrolysis by LPL (and possibly also hepatic lipase), transfer of TAG to other lipoproteins (e.g., HDL) via neutral lipid exchange, conversion of VLDL to lipoproteins of higher density, i.e., intermediate density lipoproteins (IDL) and LDL, as well as removal of the whole VLDL particle from plasma via interaction with hepatic and/or peripheral receptors [START_REF] Magkos | Women produce fewer but triglyceride-richer very low-density lipoproteins than men[END_REF]. Our kinetic data do not allow for distinguishing between these routes. Previous studies have shown that a single, prolonged bout of moderate-intensity aerobic exercise (90-120 min at 60% of VO 2 peak) enhances the efficiency of VLDL-TAG removal from plasma without affecting hepatic VLDL-TAG secretion [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Tsekouras | A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men[END_REF] and reduces the hepatic secretion of VLDL-apoB-100 [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF], indicating that the liver after exercise secretes fewer but TAG-richer (and therefore possibly also larger) VLDL particles [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF]. This is consistent with our observation that resistance exercise increased the ratio of VLDL-TAG to VLDL-apoB-100 concentrations. There is evidence from several in vivo studies in humans [START_REF] Magkos | Women produce fewer but triglyceride-richer very low-density lipoproteins than men[END_REF][START_REF] Streja | The metabolic heterogeneity of human very low density lipoprotein triglyceride[END_REF] and animals [START_REF] Streja | Triglyceride removal from very low density lipoproteins in vivo as a function of their triglyceride content[END_REF][START_REF] Verschoor | Glucose and fructose feeding lead to alterations in structure and function of very low density lipoproteins[END_REF] suggesting that the removal of TAG from the core of TAG-rich, large VLDL particles is more efficient than TAG removal from TAG-poor, small VLDL, possibly because increasing TAG content (and size) of lipoprotein particles enhances their susceptibility to hydrolysis by LPL [START_REF] Fisher | Human triacylglycerol-rich lipoprotein subfractions as substrates for lipoprotein lipase[END_REF]. We observed a significant decrease in VLDL-apoB-100 concentration after resistance exercise compared with rest, which may have resulted from reduced hepatic secretion of VLDL-apoB-100 (i.e., secretion of fewer VLDL particles), as shown previously after aerobic exercise [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF]. Hence secretion of TAG-richer VLDL after resistance exercise may The above point notwithstanding, there is evidence suggesting that LPL mRNA, protein mass and activity in skeletal muscle, but not adipose tissue, increase transiently within 4-8 h after exercise cessation and remain elevated for some 16-20 h postexercise [40][41][START_REF] Kiens | Effects of insulin and exercise on muscle lipoprotein lipase activity in man and its relation to insulin action[END_REF] or even longer [START_REF] Kiens | Utilization of skeletal muscle triacylglycerol during postexercise recovery in humans[END_REF]. This coincides with the time frame of our measurements. Elevated skeletal muscle LPL activity after exercise may therefore account for at least some of the increase in VLDL-TAG removal from the circulation we observed after a single bout of whole-body resistance exercise, by augmenting VLDL-TG clearance across the previously exercised muscles [START_REF] Kiens | Lipoprotein metabolism influenced by training-induced changes in human skeletal muscle[END_REF]10]. It has been estimated that whole-body skeletal muscle LPL activity accounts for some 10% of whole-body TAG clearance (whole-body adipose tissue LPL activity accounts for ~5%) in fasting humans at rest, but may account for as much as 50% of the clearance of exogenously infused TAG [START_REF] Seip | Skeletal muscle lipoprotein lipase: molecular regulation and physiological effects in relation to exercise[END_REF]. A single endurance exercise bout may increase skeletal muscle LPL activity by ~70% late into recovery (~18 h after exercise cessation) [START_REF] Kiens | Utilization of skeletal muscle triacylglycerol during postexercise recovery in humans[END_REF], although it does not enhance basal skeletal muscle (or adipose tissue) blood flow at that time [10]. Dynamic knee extension exercise also increases skeletal muscle LPL activity and the clearance of VLDL-TAG across the exercised muscles at rest [START_REF] Kiens | Lipoprotein metabolism influenced by training-induced changes in human skeletal muscle[END_REF][START_REF] Kiens | Effects of insulin and exercise on muscle lipoprotein lipase activity in man and its relation to insulin action[END_REF], but data on other types of resistance exercise is not available. Other possible routes for the accelerated VLDL-TAG removal after resistance exercise include enhanced TAG transfer from the core of VLDL to HDL in exchange for cholesterol esters, as well as faster removal of the VLDL particles themselves from the circulation. The first mechanism is unlikely, since enhanced transfer of TAG from VLDL to HDL after exercise would result in TAG-enrichment of HDL particles and thus accelerated clearance of HDL, thereby lowering HDL-cholesterol concentrations [START_REF] Lamarche | Kinetics of lipids, apolipoproteins, and cholesteryl ester transfer protein in plasma after a bicycle marathon[END_REF]. Furthermore, if anything, prolonged exercise has been shown to significantly reduce the concentration and activity of cholesterol ester transfer protein [45], i.e., the major enzyme responsible for the loss of TAG from the core of VLDL in exchange for cholesterol esters from HDL. Faster removal of VLDL particles themselves from the circulation, whether via conversion to lipoproteins of higher density such as IDL and LDL, or via direct uptake from the liver or peripheral tissues through interaction with specific receptors, is also unlikely to have contributed to the increase in whole-body clearance of VLDL-TAG after resistance exercise. Although we did not assess VLDL-apoB-100 kinetics, it has been shown 14 previously that the plasma clearance rate and the mean residence time of VLDL-apoB-100 (i.e., VLDL particles) in the circulation are not different the morning after single bouts of moderateintensity endurance exercise lasting 1-2 h or equivalent periods of rest [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Magkos | A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men[END_REF].

We observed that prior resistance exercise did not affect the rate of VLDL-TAG secretion from the liver. This is consistent with all previous studies examining the effects of endurance exercise on TAG metabolism in the postabsorptive state, which found no differences in hepatic VLDL-TAG secretion the morning after exercise or rest [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Tsekouras | A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men[END_REF][START_REF] Magkos | A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men[END_REF]. Interestingly, resistance exercise significantly increased basal plasma FFA concentrations by ~35%, suggesting that plasma FFA availability was higher after exercise than rest [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Magkos | A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men[END_REF]. On the other hand, we did not observe any significant changes in glucose and insulin concentrations and whole-body insulin sensitivity

(assessed with the HOMA-IR index) after resistance exercise, in accordance with previous reports [15,[START_REF] Burns | A single session of resistance exercise does not reduce postprandial lipaemia[END_REF][START_REF] Zafeiridis | Effects of low-and high-volume resistance exercise on postprandial lipaemia[END_REF]. Insulin and the availability of plasma FFA are considered the major regulatory determinants of basal VLDL-TAG secretion by the liver [START_REF] Lewis | Fatty acid regulation of very low density lipoprotein production[END_REF][START_REF] Gibbons | The roles of insulin and fatty acids in the regulation of hepatic very-low-density lipoprotein assembly[END_REF], but several recent studies provide no evidence in support of this notion under various physiological conditions in humans in vivo [START_REF] Magkos | A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men[END_REF]49,[START_REF] Gormsen | Energy expenditure, insulin, and VLDL-triglyceride production in humans[END_REF]. The fate of the excess FFA after resistance exercise is uncertain.

Some may be directed towards storage to replenish tissue TAG stores that were likely depleted by prior resistance exercise [START_REF] Koopman | Intramyocellular lipid and glycogen content are reduced following resistance exercise in untrained healthy males[END_REF]. Our data suggest that at least some of the additional FFA are oxidized in the liver, as evidenced by the almost doubling of serum beta-hydroxybutyrate concentrations the morning after exercise than rest. This may have actually prevented an increase in hepatic VLDL-TAG secretion after exercise, by limiting the intrahepatic availability of fatty acids (which would be greater after exercise than rest) for TAG synthesis and secretion.

Moreover, we observed a non-significant ~20% increase in whole-body fat oxidation rate after exercise, which did not reach statistical significance perhaps due to type II error [15,[START_REF] Jamurtas | The effects of a single bout of exercise on resting energy expenditure and respiratory exchange ratio[END_REF]; hence some of the excess FFA after exercise may be oxidized in peripheral tissues as well. Still, exercise-induced changes in total plasma TAG concentration [53] and VLDL-TAG concentration and kinetics [START_REF] Tsekouras | A single bout of brisk walking increases basal very low-density lipoprotein triacylglycerol clearance in young men[END_REF] are independent of those in basal whole-body fat oxidation. Overall, our findings suggest that the morning after a single bout of resistance exercise, basal plasma FFA availability is not the primary determinant of hepatic VLDL-TAG secretion, consistent with observations made previously following endurance exercise [START_REF] Magkos | Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men[END_REF][START_REF] Magkos | A single 1-h bout of evening exercise increases basal FFA flux without affecting VLDL-triglyceride and VLDL-apolipoprotein B-100 kinetics in untrained lean men[END_REF]. Nonetheless, results from the present study support the premise that resistance exercise may be beneficial for the management of hypertriacylglycerolemia.

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  Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T

5 Clinical

 5 such a difference is not likely of physiological importance. Calibration of the O 2 and CO 2 was performed once before the start of the exercise bout and at 45 minutes during the 2-minute resting period. For the time-matched resting trial, participants were requested to refrain from any form of physical activity and remain rested at home. Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T Stable isotope infusion The next morning, volunteers arrived at the laboratory with minimal physical activity and after having fasted overnight. One catheter was inserted into a forearm vein to administer stable isotope labeled tracers and a second catheter was inserted into a contralateral hand vein for blood sampling. The latter was kept warm with a heating pad. Catheters were flushed with 0.9% NaCl solution after each blood sampling. Subjects were allowed to relax and get used to the catheters for approximately 1 hour. At 14 ± 1 h after completion of the exercise trial or the equivalent period of rest (time = 0), a baseline blood sample was taken to determine fasting plasma and serum metabolite concentrations and background tracer-to-tracee ratio (TTR) of glycerol in VLDL-TAG. A bolus of [1,1,2,3,3-2 H 5 ]glycerol (75 μmol/kg body wt; Goss Scientific Instruments Ltd., Essex, UK), dissolved in 0.9% NaCl solution, was then administered through the catheter in the forearm vein, and blood samples were obtained at 15 min after tracer injection and then every hour for 6 h, to determine glycerol TTR in VLDL-TAG. Indirect calorimetry was performed using a ventilated canopy (Sensormedics, Vmax229D, Yorba Linda, CA) for 15 min before catheter insertion and every hour after tracer injection. The first 5 min served as a habituation period. Oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) were measured for the remaining 10 min to estimate resting metabolic rate (RMR) [22] and wholebody fat oxidation rate [23]. For the entire duration of the studies, volunteers remained fasted in the laboratory in a sitting position. Water consumption was allowed ad libitum.

( 7 Clinical

 7 μU/ml) × fasting plasma glucose (mmol/l) / 22.5[START_REF] Matthews | Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man[END_REF]. Paired samples for each volunteer were analyzed in the same batch. For each trial, a small aliquot of baseline blood samples was used for determination, in triplicates, of haematocrit and haemoglobin concentration by microhaematocrit and cyanomethaemoglobin assays, respectively (Boehringer Mannheim Diagnostics, Mannheim, Germany). Changes in plasma volume between rest and exercise trials were assessed by using the Dill and Costill equation[START_REF] Dill | Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration[END_REF]. Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T

  transferred into Quick Seal Centrifuge Polyallomer Tubes (Beckman Instruments Inc., Palo Alto, CA), overlaid with a NaCl/EDTA solution (d = 1.006 g/ml) and spun for 3 h at 90000 rpm at 4°C, in an Optima TLX ultracentrifuge equipped with the fixed-angle TLN-100 rotor (Beckman Instruments Inc., Palo Alto, CA). The top layer, containing the VLDL, was removed and collected quantitatively by tube slicing (CentriTube slicer, Beckman Instruments Inc., Palo Alto, CA), and stored at -80°C until analyses. The concentration of VLDL-TAG was measured by using an enzymocolorimetric assay (Alfa Wassermann Diagnostics Technologies, West Caldwell, NJ) and the concentration of VLDL-apolipoprotein B-100 (VLDL-apoB-100) was measured immediately in fresh samples by using an immunoturbidimetric assay (Randox Laboratories Ltd., Antrim, UK) on an automated analyzer (ACE Schiapparelli Biosystems, Fairfield, NI).

( 8 Clinical

 8 SpeedVac, ThermoSavant, Holbrook, NY), before isolating VLDL-TAG by thin layer chromatography (TLC). Dried samples were resuspended in chloroform : methanol (3:1), spotted on a LK6D silica gel plate (60 Å silica gel, 250 μm layer thickness; Whatman, Maidstone, UK), and developed with heptane : diethyl ether : formic acid (80:20:2) in an enclosed developing chamber. The TAG band was visualized with 0.01% rhodamine 6G in the TLC lanes, scraped off the TLC plate, and transferred into 13×160 mm test tubes. TAG were extracted from the silica gel with chloroform : methanol (3:1) and dried under vacuum. The dried samples were hydrolyzed by resuspension in 10% acetyl chloride in methanol and incubation for 30 min at 70°C, and then dried under vacuum. Glycerol was derivatized with heptafluorobutyric (HFB) anhydride (30 min incubation at 70°C) and samples were dried under vacuum. Dry HFB-glycerol was dissolved in heptane and stored at -40°C. The TTR of glycerol in VLDL-TAG was determined by gas-chromatography / mass-spectrometry (GC/MS; MSD 5973 system, Hewlett-Packard, Palo Alto, CA), by selectively monitoring the ions at mass to charge ratios (m/z) 467 and 472[START_REF] Patterson | Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover[END_REF]. Calibration curve for standards with known isotopic enrichment was used. Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T

Clinical

  Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T concentrations was significantly increased by exercise (Table

Clinical

  Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T exercise [11-14]. It has long been recognized [32] that the hypotriacylglycerolemic effect of endurance exercise manifests acutely, ~12 hours after the cessation of a single exercise bout, lasts for 1-2 days and does not result from metabolic adaptations to repeated exercise sessions (i.e., training) [33,34]. Resistance/weight training also favourably modifies several risk factors

  macronutrient composition of the diet, including the last meal (dinner) consumed after the 12 Clinical Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T completion of the exercise and resting sessions. Hence subjects were in a negative energy balance during the exercise compared with the resting trial, by the net energy expenditure of the exercise session (i.e., by ~1.2 MJ). And, we did observe a significant reduction in fasting plasma TAG and VLDL-TAG concentrations the morning after exercise than rest; the main purpose of our study was to describe the mechanisms for the hypotriacylglycerolemic effect of acute resistance exercise.

  underlie the augmented plasma clearance rate of VLDL-TAG via enhanced whole-body LPL-13 Clinical Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T mediated lipolysis; this mechanism does not presuppose any changes in LPL activity of tissues after exercise.

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  Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T

15

  Clinical Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T 16 In summary, a single bout of strenuous resistance exercise in the evening lowers total plasma and VLDL-TAG concentrations the next morning in healthy, normolipidemic, non-obese, untrained young men. Augmented VLDL-TAG removal from the circulation is the mechanism responsible for the hypotriacylglycerolemic effect of acute resistance exercise, without any changes in VLDL-TAG secretion from the liver. The effects of resistance and endurance exercise on basal VLDL-TAG metabolism appear thus to be qualitatively and quantitatively similar. It is currently not known whether resistance exercise similarly affects VLDL-TAG metabolism in other population groups (e.g., in women), or in high-risk individuals such as the obese patients or those suffering from hyperlipidemia. These questions should be addressed in future investigations.

Figure 1 .

 1 Figure 1. Very low-density lipoprotein-triacylglycerol (VLDL-TAG) concentration in plasmathroughout the 6-h sampling period the morning after a single evening bout of resistance exercise and an equivalent period of rest. Values are means ± SEM. There is a significant effect of trial (exercise versus rest, P = 0.03), but no effect of time (P = 0.29), or an interaction between trial and time (P = 0.89).

Figure 2 .

 2 Figure 2. Time-course of tracer-to-tracee ratio (TTR) of glycerol in very low-density lipoprotein-triacylglycerol (VLDL-TAG) (a) and ln-transformed TTR data (b) throughout the 6-h sampling period the morning after a single evening bout of resistance exercise and an equivalent period of rest. Values are means ± SEM.

Figure 3 .

 3 Figure 3. Hepatic very low-density lipoprotein-triacylglycerol (VLDL-TAG) secretion rate (a) and whole-body VLDL-TAG plasma clearance rate (b) the morning after a single evening bout of resistance exercise and an equivalent period of rest. Values are means ± SEM. * Significantly different from rest (P = 0.004).

ClinicalFigure 1 Time 26 Clinical 27 Clinical

 12627 Figure 1

28 Clinical

 28 Figure 3

  . It was assumed that VLDL-TAG volume of distribution is equal to PV since VLDL particles are essentially restricted in the plasma compartment[30]. The rate of whole-body VLDL-TAG plasma clearance (ml/min), which is an index of the efficiency of VLDL-TAG removal from the circulation, was calculated by dividing the rate of VLDL-TAG disappearance from plasma

	relative VO 2 peak were 3.4 ± 0.3 l/min and 42.8 ± 2.8 ml/kg•min, respectively. Dietary energy
	intake during the 3 days preceding each trial averaged 8.2 ± 0.9 MJ/day (48 ± 4% from
	carbohydrate, 36 ± 6% from fat, and 16 ± 2% from protein), and the last meal (dinner) consumed
	after the completion of the exercise and resting sessions provided 3.9 ± 0.5 MJ (119 ± 1 g
	carbohydrate, 34 ± 7 g fat, and 35 ± 7 g protein). Average oxygen consumption during the
	resistance exercise session was 10.9 ± 0.8 ml/kg•min, at an average heart rate of 125 ± 5 bpm

(μmol/min), which equals VLDL-TAG secretion rate under steady state conditions, by the concentration of VLDL-TAG in plasma (μmol/ml). The mean residence time of VLDL-TAG in the circulation (min) was calculated as 60 / FCR.

Statistical analysis

Data were analyzed using SPSS 11.0 (SPSS Inc., Chicago, IL). All variables were normally distributed according to the Shapiro-Wilks test; hence comparisons between rest and exercise were performed by using parametric procedures, and results are presented as means ± SEM. The Student's t-test for paired samples was used to evaluate metabolic and kinetic parameters between rest and exercise. Analysis of covariance for repeated measures was performed to test the stability of total plasma and VLDL-TAG concentrations and VO 2 throughout the 6-h kinetic study for both trials, taking into account inter-correlation between time points. Covariates as potential cofounders were, age, fat-free mass, body mass index and VO 2 peak. Statistical significance was set at P ≤ 0.05. Based on a previous reproducibility study, a sample size of 8 would allow for detecting physiologically significant differences in VLDL-TAG secretion and clearance rates of ≥20-25% in magnitude, at alpha = 5% and with 80% power [31]. 9 Clinical Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T RESULTS Body composition, VO 2 peak, dietary intake and exercise energy expenditure Average fat mass and fat-free mass were 14 ± 1 kg and 64 ± 3 kg, respectively. Absolute and (including the resting intervals between the exercise sets). Mean RQ during exercise averaged 1.02 ± 0.01. The gross energy expenditure of exercise was 1.63 ± 0.17 MJ, whereas exercise net energy expenditure (i.e., above resting values) was 1.19 ± 0.15 MJ. Mean work production for each exercise and total work production of the entire exercise bout, measured by the isokinetic dynamometer, are shown in Table

Table 1 .

 1 Work production for each exercise and total work production of the entire resistance exercise bout performed on the isokinetic dynamometer

	Exercise	Angular velocity (degrees/sec)	Work production (joules)
	Leg press	120	8444 ± 654
	Leg pull	120	1860 ± 178
	Shoulder press	180	1786 ± 287
	Chin ups	180	4499 ± 493
	Knee extension	120	9417 ± 908
	Knee curl	120	5664 ± 977
	Chest press	150	3449 ± 335
	Upright row	150	2181 ± 202
	Hip flexion (horizontal position)	120	5944 ± 1096
	Hip extension (horizontal position)	120	4097 ± 317
	Shoulder abduction	120	2882 ± 314
	Shoulder adduction	120	9102 ± 876
	Total work production		59326 ± 986
	Values are means ± SEM. Work production for each exercise is presented as the sum of left and
	right sides for all sets.

S t a g e 2 ( a ) P O S T -P R I N T

Table 2 .

 2 Fasting metabolite concentrations the morning after a single evening bout of resistance exercise and an equivalent period of rest

	Rest	Exercise	P-value

HOMA-IR, homeostasis model assessment of insulin resistance; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TAG, triacylglycerol; VLDL, very low-density lipoprotein; apoB-100, apolipoprotein B-100.

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

ACKNOWLEDGEMENTS

We would like to thank all participants that took part in the present study, which was funded by the Graduate Program of Harokopio University, Athens, Greece. 17 Clinical Science Immediate Publication. Published on 13 Jun 2008 as manuscript CS20080078 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080078 S t a g e 2 ( a ) P O S T -P R I N T


M Loredana Marcovecchio

Barry Widmer

Professor David B Dunger

R Neil Dalton

Effect of acute variations of insulin and glucose on plasma concentrations of asymmetric dimethylarginine in young people with type 1 diabetes

Keywords: Asymmetric dimethylarginine, insulin and glucose asymmetric dimethylarginine, symmetric dimethylarginine, type 1 diabetes, insulin, glycemia, cardiovascular disease

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is considered a major risk factor for cardiovascular disease and progression of renal disease. In the present study we aim to investigate the effect of acute variations in plasma glucose and insulin on plasma ADMA levels in young people with type 1 diabetes (T1D).

Fifteen young patients (10 males) with T1D, median age 18.3 (13.2-24.4) years, HbA1c 9% (6.4-13.6), underwent an overnight (18:00-08:00 h) variable insulin infusion for euglycemia, followed by a hyperinsulinemic euglycemic clamp (08:00-12:00 h). Blood samples were collected every 15 minutes for determination of ADMA, symmetric dimethylarginine (SDMA), valine, phenylalanine, arginine, creatinine and glucose. Insulin levels were assessed every 30 minutes.

During the overnight period, glucose levels increased following the evening meal. In response to the protein intake there was a significant increase in ADMA, arginine, valine, phenylalanine and creatinine. During the remaining part of the night, glucose levels progressively decreased reaching 5 mmol/l by 04:00. ADMA and SDMA did not change significantly. During the hyperinsulinemic clamp a significant fall in ADMA was observed, from 0.468±0.056 to 0.364±0.050µmol/l (p<0.001). A significant fall was also found in SDMA, valine, phenylalanine, arginine and the ADMA/SDMA ratio (all p<0.001), but not in creatinine levels. No correlation was found between insulin sensitivity and ADMA.

We conclude that acute changes in glycemia do not significantly affect plasma ADMA levels whereas infusion of insulin significantly reduces ADMA suggesting an important role of insulin in the regulation of this cardiovascular risk factor.

INTRODUCTION

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is now considered to be an important biomarker in the assessment of cardiovascular risk [START_REF] Siroen | The clinical significance of asymmetric dimethylarginine[END_REF][START_REF] Böger | Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond[END_REF]. ADMA and its stereoisomer, symmetric dimethylarginine (SDMA), are synthesised during post-translational modification of L-arginine residues by the action of protein-argininemethyltransferases and subsequently released during proteolysis [START_REF] Kakimoto | Isolation and identification of N G , N G -and N G , N' G -dimethyl-arginine, N-epsilon-mono-, di-, and trimethyllysine, and glucosylgalactosyl-and galactosyl-delta-hydroxylysine from human urine[END_REF][START_REF] Mcdermott | Studies on the catabolism of N G -methylarginine[END_REF]. Early reports of increased ADMA concentrations in chronic renal failure patients on haemodialysis [START_REF] Vallance | Accumulation of an endogenous inhibitor of NO synthesis in chronic renal failure[END_REF][START_REF] Wahbi | Dimethlarginines in chronic renal failure[END_REF] and the significant decline in ADMA concentrations post-dialysis suggested an important role of the kidney in the elimination of ADMA. However, it is now recognised that approximately 80-90% of the ADMA released is metabolised to citrulline and dimethylamine by dimethylarginine dimethylaminohydrolase (DDAH) [START_REF] Macallister | Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Tran | The DDAH/ADMA/NOS pathway[END_REF]. DDAH is expressed in a wide range of tissues [START_REF] Macallister | Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Tran | The DDAH/ADMA/NOS pathway[END_REF][START_REF] Vallance | Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway[END_REF], but the increase in plasma ADMA observed in liver failure and its rapid normalisation post liver transplantation [START_REF] Mookerjee | Inflammation is an important determinant of levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in acute liver failure[END_REF][START_REF] Siroen | The transplanted liver graft is capable of clearing asymmetric dimethylarginine[END_REF] demonstrate the importance of the liver in ADMA metabolism and in maintaining plasma ADMA concentrations. Increased plasma ADMA is consistently linked with poor clinical outcomes [START_REF] Schnabel | Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the Athero Gene Study[END_REF][START_REF] Schulze | Asymmetric dimethylarginine is an independent risk factor for coronary heart disease: results from the multicenter Coronary Artery Risk Determination investigating the Influence of ADMA Concentration (CARDIAC) study[END_REF][START_REF] Kryzanowska | Asymmetric dimethylarginine predicts cardiovascular events in patients with type 2 diabetes[END_REF][START_REF] Nijveldt | Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality[END_REF][START_REF] Mookerjee | Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis[END_REF][START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF][START_REF] Fliser | Asymmetric dimethylarginine and progression of chronic kidney disease: the mild to moderate kidney disease study[END_REF][START_REF] Ravani | Asymmetrical dimethylarginine predicts progression to dialysis and death in patients with chronic kidney disease: a competing risks modeling approach[END_REF], being an independent risk factor for cardiovascular disease (CVD) [START_REF] Schnabel | Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the Athero Gene Study[END_REF][START_REF] Schulze | Asymmetric dimethylarginine is an independent risk factor for coronary heart disease: results from the multicenter Coronary Artery Risk Determination investigating the Influence of ADMA Concentration (CARDIAC) study[END_REF][START_REF] Kryzanowska | Asymmetric dimethylarginine predicts cardiovascular events in patients with type 2 diabetes[END_REF] and mortality [START_REF] Nijveldt | Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality[END_REF][START_REF] Mookerjee | Increasing dimethylarginine levels are associated with adverse clinical outcome in severe alcoholic hepatitis[END_REF][START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF] and a predictor of progression of renal disease [START_REF] Fliser | Asymmetric dimethylarginine and progression of chronic kidney disease: the mild to moderate kidney disease study[END_REF][START_REF] Ravani | Asymmetrical dimethylarginine predicts progression to dialysis and death in patients with chronic kidney disease: a competing risks modeling approach[END_REF]. Consequently, it is essential that we understand the basic mechanisms resulting in increased plasma ADMA concentrations to enable the development of therapeutic strategies.

In non diabetic subjects there is a significant correlation between plasma ADMA concentrations and insulin resistance [START_REF] Stuhlinger | Relationship between insulin resistance and an endogeneous nitric oxide synthase inhibitor[END_REF][START_REF] Marliss | Elevations of plasma methylarginines in obesity and ageing are related to insulin sensitivity and rates of protein turnover[END_REF][START_REF] Mclaughlin | Plasma asymmetric dimethylarginine concentrations are elevated in obese insulin-resistant women and fall with weight loss[END_REF]. In contrast, increased ADMA in women with gestational diabetes is not associated with insulin resistance [START_REF] Mittermayer | Circulating concentrations of asymmetrical dimethyl-L-arginine are increased in women with previous gestational diabetes[END_REF]. However, it is possible that in insulin resistance states the observations may be confounded by increased glucose or differences in insulin levels. Incubation of vascular cells with glucose resulted in a decline in DDAH activity and an accumulation of ADMA [START_REF] Sorrenti | High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells[END_REF]. In streptozotocin-induced diabetic rats, plasma ADMA was increased as a result of reduced DDAH activity [START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF]. In contrast, in other experimental studies, no changes in ADMA [START_REF] Palm | Reduced nitric oxide concentration in the renal cortex of streptozotocin-induced diabetic rats: effects on renal oxygenation and microcirculation[END_REF], or even a decline in its concentrations, were associated with hyperglycemia [START_REF] Palm | Reduced nitric oxide in diabetic kidney due to increased hepatic arginine metabolism: implications for renomedullary oxygen availability[END_REF]. The latter finding could be related to an activation of hepatic DDAH by high glucose levels, effect recently reported in diabetic rats [START_REF] Chobanyan | GC-MS assay for hepatic DDAH activity in diabetic and non-diabetic rats by measuring dimethylamine (DMA) formed from asymmetric dimethylarginine (ADMA): Evaluation of the importance of S-nitrosothiols as inhibitors of DDAH activity in vitro and in vivo in humans[END_REF] and which differs from previous findings of an inhibition of DDAH by hyperglycemia [START_REF] Sorrenti | High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells[END_REF][START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF]. In addition, in vivo, no change in ADMA was measured following a saccharose challenge [START_REF] Chobanyan | GC-MS assay for hepatic DDAH activity in diabetic and non-diabetic rats by measuring dimethylamine (DMA) formed from asymmetric dimethylarginine (ADMA): Evaluation of the importance of S-nitrosothiols as inhibitors of DDAH activity in vitro and in vivo in humans[END_REF].

In subjects with type 1 diabetes (T1D) plasma ADMA is increased [START_REF] Tarnow | Elevated plasma asymmetric dimethylarginine as a marker of cardiovascular morbidity in early diabetic nephropathy in type 1 diabetes[END_REF][START_REF] Eid | Increased levels of asymmetric dimethylarginine in populations at risk for atherosclerotic disease. Effect of pravastatin[END_REF][START_REF] Altinova | Uncomplicated ype 1 diabetes is associated with increased asymmetric dimethylarginine concentrations[END_REF], whereas in type 2 diabetes is reduced and negatively correlated with HbA1c [START_REF] Paiva | Plasma concentrations of asymmetric-dimethyl-arginine in type 2 diabetes is associated with glycemic control and glomerular filtration rate but not with risk factors of vasculopathy[END_REF]. In vitro, insulin reverses the accumulation of ADMA in endothelial cells incubated with tumor necrosis factor-(TNF)alfa by increasing DDAH activity [START_REF] Eid | Insulin and adiponectin inhibit the TNFα-induced ADMA accumulation in human endothelial cells. The role of DDAH[END_REF] and, in vivo, ADMA is reduced during acute hyperinsulinemia in non-diabetic subjects [START_REF] Eid | Decreased levels of asymmetric dimethylarginine during acute hyperinsulinemia[END_REF].

The predictive value of plasma ADMA concentrations is of fundamental importance in subjects with T1D, where the risk of premature heart disease and coronary events remains unacceptably high [START_REF] Orchard | Type 1 diabetes and coronary artery disease[END_REF]. In the diabetic population, chronic elevations in ADMA levels have been suggested as a mediator of endothelial dysfunction and the associated risk of developing CVD [START_REF] Kryzanowska | Asymmetric dimethylarginine predicts cardiovascular events in patients with type 2 diabetes[END_REF][START_REF] Lajer | Plasma concentration of asymmetric dimethylarginine (ADMA) predicts cardiovascular morbidity and mortality in type 1 diabetic patients with diabetic nephropathy[END_REF]. However, the value of any 'chronic marker' of CVD risk is dependent on an understanding of factors, which acutely affect changes in plasma levels. With respect to T1D, as plasma insulin and glucose vary over any 24-hour period, it is essential to understand how their acute fluctuations might modulate plasma ADMA levels.

The aim of the present study was to investigate acute changes in ADMA, in a wellcharacterized group of adolescents and young adults with T1D in relation to glucose and insulin levels. Furthermore, we aimed to assess changes in SDMA, arginine and the amino acids valine and phenylalanine in order to determine the relationship between dimethylarginine metabolites and other insulin sensitive measures of protein catabolism.

MATERIALS AND METHODS

Study population.

Plasma samples from 15 subjects (10 males and 5 females) with T1D collected during a previous study [START_REF] Saukkonen | Dose-dependent effects of recombinant human insulin like growth factor (IGF)-I/IGF binding protein-3 complex on overnight growth hormone secretion and insulin sensitivity in type 1 diabetes[END_REF] and stored at -80 o C were used in this study. The aim of the original study was to determine the effect of IGF-I/IGFBP-3 complex administration on insulin sensitivity in patients with T1D. Subjects were investigated after a 2-day course of IGF-I/IGFBP-3 complex or of placebo. In the present study only samples collected after placebo treatment were used to assess 15-minute plasma levels of ADMA, SDMA, arginine, creatinine, valine and phenylalanine and 30-minute levels of plasma insulin.

Inclusion criteria were: T1D of at least 2-year duration or C-peptide negative, age between 13-25 yr, puberty (Tanner) at least stage II, treatment with at least two insulin injections per day and normal renal and liver function. Exclusion criteria were: body mass index (BMI) >30 Kg/m 2 , untreated hypothyroidism, chronic illness, pregnancy, malignancy or recurrent episodes of severe unexplained hypoglycemia. All subjects underwent an overnight (18:00-08:00 h) variable insulin infusion to establish and maintain euglycemia (5 mmol/l), followed by a 4-hour 2-step hyperinsulinemic euglycemic clamp.

The study was approved by the Cambridge and Northampton Local Research Ethics

Committees, and written informed consent was obtained from all subjects and/or from their parents. All overnight studies were performed in Cambridge in the Addenbrooke's Clinical Research Centre, Wellcome Trust Clinical Research Facility.

Study protocol.

Long-and intermediate-acting insulin were withdrawn and substituted with regular soluble insulin injections. Sampling and infusion cannulae were inserted, an intravenous insulin infusion started, and subjects allowed an unrestricted evening meal. The insulin infusion was continued overnight and adjusted in response to 15 min blood glucose determinations in order to achieve glucose concentrations around 5 mmol/l by 04:00. During the period 04:00 to 08:00, the steady-state period, glucose concentrations were maintained around, and did not significantly differ from 5 mmol/l. Mean plasma free insulin and insulin requirement to maintain euglycemia (mean insulin infusion per kilogram of free-fat mass (mU/Kg FFM))

were both used to assess insulin sensitivity during the steady-state period. At 08:00 a.m., a 4h, two-step hyperinsulinemic euglycemic clamp was performed. Between 08:00-10:00 h, a lowdose insulin infusion (step 1: insulin bolus, 2.8 mU/Kg; infusion, 0.6 mU/Kg/min) was administered and euglycemia maintained by an infusion of 20% dextrose. Between 10:00-12:00 h, the insulin infusion rate was increased (step 2: insulin bolus, 7.0 mU/Kg; insulin infusion, 1.5 mU/Kg/min) and the infusion of 20% dextrose increased, as necessary, to maintain euglycemia. Blood glucose measurements were performed every 5 min.

M-value was calculated as mean glucose infusion rate per kg of FFM (mg/Kg FFM) during the steady-state periods of step 1 (09:30-10:00 h) and of step 2 (11:30-12:00 h) and insulin sensitivity was calculated by dividing M-value by the steady-state insulin levels and expressed as M/I. Plasma samples were collected every 15 min for the measurement of ADMA, SDMA, creatinine, arginine, valine and phenylalanine. Plasma insulin levels were measured in samples collected every 30 minutes.

Anthropometry

On arrival height and weight were determined while the subjects stood in light clothing with a wall-mounted stadiometer and an electronic scale, respectively. BMI was calculated as weight divided by the square of the height. Body fat mass and percentage of body fat were estimated using a bioelectrical impedance monitor (Biostat 1500, Isle of Man, UK). FFM was calculated as total body mass subtracted by the amount of fat mass as determined by bioimpedance.

Biochemical analyses

Plasma ADMA, SDMA, creatinine, arginine, valine and phenylalanine were measured by stable isotope dilution electrospray mass spectrometry-mass spectrometry (MSMS). Plasma (50 µL) was mixed with 50 µL of deionised water containing 50 pmol 2 H 6 -ADMA, 50 pmol Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

3.1% and arginine 107 µmol/l, 2.5%; 208 µmol/l, 1.7%; 465 µmol/l, 2.8%. Inter-assay variation (n=34) was: valine 194 µmol/l, 2.6% and phenylalanine 64.0 µmol/l, 2.6%.

Blood glucose concentrations were measured using YSI model 2300 stat plus analyzer (YSI, Lynchford House, Farnborough, Hampshire, UK). The intraassay coefficient of variation (CV) at 4.1 mmol/l was 1.5%. The equivalent interassay CV at this glucose concentration was 2.8% and was 1.7% at 14.1 mmol/l. Plasma free insulin concentrations were measured using an ELISA (Dako Ldt., Denmark House, Angel Drove, Ely, Cambridgeshire, UK) according to the manufacturer's instructions.

Intraassay imprecision (CV) was 4.3% at 82 pmol/l (14 mU/l), 3% at 402 pmol/l (67 mU/l), and 5.7% at 907 pmol/l (151 mU/l). Equivalent interassay imprecision was 4.3, 5.1 and 5.4%, respectively.

Statistical analysis

Statistical analysis was performed using SPSS version 11.5 software for Windows (SPSS Inc., Chicago, USA). Data are expressed as mean ± SD (or range) and median (range). P < 0.05 was considered significant for all data analysis. ANOVA for repeated measurement was used to assess changes in variables during the overnight period (18:00-04:00) and during the hyperinsulinemic clamp. Differences in variables between specific time points were analysed by paired t-test. Pearson correlation analysis was used to establish associations between variables of interest.

RESULTS

The clinical characteristics of the study population are summarized in Table 1.

Overnight insulin infusion for euglycemia (18:00-04:00 h) (Table 2).

In response to the evening meal, median plasma glucose increased from 11.0 (4.0-20.7) mmol/l to a peak of 13.6 (9.1-23.2) mmol/l at 20:15 (p=0.17) before a progressive decline to 5.2 (4.3-6.7) mmol/l by 04:00 (Figure 1). Median plasma insulin increased from 99 (62-511) pmol/l to 346 (120-863) pmol/l and was 73 (62-181) pmol/l by 04:00. Plasma ADMA increased from 0.446±0.073 µmol/l to a peak of 0.493±0.075 µmol/l by 20:30 (p<0.001) but then remained constant through to 04:00 (Figure 1). Significant increases were observed for arginine, 36.6±10.8 µmol/l to peak, 75.9±33.8 µmol/l at 00:00 (p=0.001), valine, 217±37 µmol/l to peak, 283±67 µmol/l at 22.30 (p=0.001), phenylalanine, 65.2±7.0 µmol/l to peak, 88.8±12.7 µmol/l at 22.30 (p<0.001), and creatinine, 59.2±9.7 µmol/l to peak, 63.0±9.3 µmol/l at 21.30 (p=0.05). All, except creatinine, declined significantly through to 04:00 with phenylalanine at this time being significantly lower, 58.8±10.9 µmol/l (p=0.002) than at 18:00. Arginine was still significantly higher at 04:00, 52.1±12. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

to 18:00. Plasma SDMA and the ADMA/SDMA ratio remained constant over the period 18:00 to 04:00 Overnight steady-state period (04:00-08:00 h) (Table 3) Euglycemia was maintained during this period. No significant differences were found in plasma ADMA, SDMA, and creatinine between 04:00 and 08:00 h, whereas valine and phenylalanine were lower at 08:00 h. Insulin sensitivity, assessed either by mean plasma insulin levels or insulin requirement for euglycemia, did not correlate with plasma ADMA, SDMA, valine, phenylalanine, arginine or creatinine (all p>0.05).

Euglycemic hyperinsulinemic clamp (08:00-12:00 h)(Table 4)

Euglycemia was maintained during this period. Plasma free insulin increased significantly from 104 [62-296] to 465 [228-574] pmol/l by the end of the clamp. In response (Figure 2), there were significant (p<0.001) declines in plasma ADMA (22%), 0.468±0.056 to 0.364±0.050 µmol/l, SDMA (9%), 0.414±0.046 to 0.373±0.040 µmol/l, the ADMA/SDMA ratio, 1.11 [0.92-1.49] to 0.95 [0.77-1.31], arginine (23%), 55.7±13.7 to 42.9±10.7 µmol/l, valine (33%), 213±35 to 142±21 µmol/l, and phenylalanine (29%), 52.9±9.8 to 37.4±8.4 µmol/l. In contrast, plasma creatinine did not change.

The pattern of decline of valine and phenylalanine was similar to that of ADMA and SDMA (Figure 2). A significant strong correlation was found between mean plasma ADMA concentrations at each time point during the clamp and mean valine (r=0.98; p<0.0001) as well as phenylalanine (r=0.97; p<0.0001) at corresponding time points Insulin sensitivity, assessed as M/I ratio was predictably higher during step 2 than step 1 (0.21±0.01 vs 0.016±0.008 mg•kg -1 •min -1 /pmol/l; p=0.024). No differences were found in the rate of decline of plasma ADMA, SDMA, arginine, valine or phenylalanine between the two steps of the clamp (data not shown). Furthermore, no correlation was found between M/I ratio and the rate of decline of plasma ADMA, SDMA, arginine, valine and phenylalanine during either step 1 or step 2.

DISCUSSION

The present study utilises samples obtained in a previous study [START_REF] Saukkonen | Dose-dependent effects of recombinant human insulin like growth factor (IGF)-I/IGF binding protein-3 complex on overnight growth hormone secretion and insulin sensitivity in type 1 diabetes[END_REF] to assess the acute effect of changes in glucose and insulin on plasma levels of ADMA in a group of young patients with T1D. The majority of studies relating plasma ADMA concentrations to physiological changes have been based on a single measurement. In part, this has been due to the technical difficulty, and resultant time and costs, associated with conventional analytical techniques.

The use of high sensitivity MSMS enables large numbers of samples to be processed economically with accuracy and precision. This study represents the first investigation of sequential, 15-minute, plasma ADMA concentrations in response to controlled, significant changes in plasma insulin and glucose levels in a group of young patients with T1D.

Plasma ADMA concentration represents the balance between the rate of production and the rate of removal [START_REF] Tran | The DDAH/ADMA/NOS pathway[END_REF][START_REF] Vallance | Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway[END_REF]. In acute circumstances the rate of production of ADMA will be, almost exclusively, dependent on the rate of proteolysis and the rate of removal will be dependent, primarily, on DDAH activity [START_REF] Tran | The DDAH/ADMA/NOS pathway[END_REF][START_REF] Vallance | Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway[END_REF]. However, the production side of the equation will also be a function of dietary intake and a significant amount of the removal of ADMA will rely on renal function [START_REF] Tran | The DDAH/ADMA/NOS pathway[END_REF][START_REF] Vallance | Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway[END_REF]. This study allows us to consider all these components, with some being better controlled than others. Throughout the study there is no significant change in glomerular filtration rate, as assessed by plasma creatinine. The increase in plasma creatinine in the early post-prandial phase, before 22:00, is to be expected following an unrestricted meal, as cooked meat contains significant quantities of creatinine, produced during cooking by dehydration of creatine, which is readily absorbed and increases plasma creatinine [START_REF] Preiss | The influence of a cooked meat meal on estimated glomerular filtration rate[END_REF].

The increases in valine, phenylalanine, and arginine are the result of dietary protein hydrolysis in the gut and absorption of the resulting amino acids. Since both ADMA and SDMA are likely to be components of dietary proteins, post-prandial increases in ADMA and SDMA might be expected. In the group as whole plasma ADMA did increase significantly, by 0.047 µmol/l. This is an interesting observation, as one might expect significant first pass metabolism of ADMA in the liver. Surprisingly, plasma SDMA did not change. Since SDMA is not metabolised and is almost exclusively removed by renal clearance [START_REF] Kielstein | Symmetric dimethylarginine (SDMA) an endogenous marker of renal function -a meta-analysis[END_REF] it might be expected to increase post-prandially. However, the general ratio of ADMA to SDMA in most tissues is approximately 5:1 [START_REF] Bulau | Quantitative assessment of arginine methylation in free versus proteinincorporated amino acids in vitro and in vivo using protein hydrolysis and highperformance liquid chromatography[END_REF] and, as ADMA only increased 0.047 µmol/l, SDMA might be expected to increase by 0.010 µmol/l; sufficiently small to be difficult to detect.

In the post-prandial phase, plasma glucose increased to a median value of 13.6 mmol/l and then declined progressively over the next 8 hours to 5 mmol/l. Despite these physiologically major changes in plasma glucose during the overnight period there was virtually no change in the mean plasma ADMA concentration. In this phase, absorption of ADMA is likely to be negligible and GFR, based on plasma creatinine levels, is constant indicating that DDAH activity would be the predominant factor in determining plasma ADMA concentrations. It might be hypothesized that the rapid post-prandial increase in plasma glucose could have inactivated DDAH and this could explain the initial post-prandial rise in ADMA, but not in SDMA, and the sustained high ADMA levels during the remaining part of the overnight period, as DDAH might require a prolonged period to re-gain its full activity. The reported inhibitory effects of glucose on DDAH activity in cells incubated with glucose [START_REF] Sorrenti | High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells[END_REF][START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF] and the protective effect of superoxide dismutase co-incubation [START_REF] Sorrenti | High glucose-mediated imbalance of nitric oxide synthase and dimethylarginine dimethylaminohydrolase expression in endothelial cells[END_REF] implies an oxidative mechanism for glucose-mediated increases in ADMA and offers compelling evidence for a Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

hyperglycemia-driven inhibitory mechanism on DDAH. However, animal studies have reported controversial results on the effect of glucose on DDAH activity. On the one hand, in streptozotocin-induced diabetic rats increased plasma ADMA and reduced DDAH activity have been associated with hyperglycemia. On the other hand, in other models of streptozotocin-induced diabetes, ADMA levels were unchanged [START_REF] Palm | Reduced nitric oxide concentration in the renal cortex of streptozotocin-induced diabetic rats: effects on renal oxygenation and microcirculation[END_REF] or even decreased [START_REF] Palm | Reduced nitric oxide in diabetic kidney due to increased hepatic arginine metabolism: implications for renomedullary oxygen availability[END_REF].

The latter phenomenon could be explained by an increased DDAH activity in response to hyperglycemia [START_REF] Chobanyan | GC-MS assay for hepatic DDAH activity in diabetic and non-diabetic rats by measuring dimethylamine (DMA) formed from asymmetric dimethylarginine (ADMA): Evaluation of the importance of S-nitrosothiols as inhibitors of DDAH activity in vitro and in vivo in humans[END_REF].

During the post-prandial period, there was a significant rise in plasma insulin levels, arising from compensatory increases in the insulin infusion rates associated with rises in glucose levels. In contrast with the significant effect of insulin during the hyperinsulinemic clamp, the higher post-prandial insulin levels seemed not to affect plasma ADMA. A possible explanation for this observation is that the influx of dietary ADMA overwhelmed systemic clearance and thus masked the insulin effect on ADMA levels.

In sharp contrast to the benign response to glycemia, euglycemic hyperinsulinemic infusion resulted in a rapid decline in plasma ADMA and SDMA. Since, SDMA is being released by proteolysis, is an end-product of metabolism cleared by the kidney [START_REF] Kielstein | Symmetric dimethylarginine (SDMA) an endogenous marker of renal function -a meta-analysis[END_REF], and there is no change in GFR, the decline is almost certainly due to reduced proteolysis. In this small study, the effect on plasma ADMA appears to be proportionately greater, resulting in a significant decline in the ADMA/SDMA ratio. This may simply reflect the higher ADMA, compared to SDMA, content of protein [START_REF] Bulau | Quantitative assessment of arginine methylation in free versus proteinincorporated amino acids in vitro and in vivo using protein hydrolysis and highperformance liquid chromatography[END_REF][START_REF] Al | N' G -dimethylarginine in renal insufficiency[END_REF], but an additional increase in DDAH activity cannot be excluded. Note that the proportional decreases in valine and phenylalanine are even greater, presumably because the insulin-dependent protein synthesis rate is increased relative to the proteolytic rate. Since dimethylarginines, in contrast to valine and phenylalanine, are not incorporated in de novo synthesis of proteins and they are produced as part of a posttranslational methylation mainly of nuclear proteins [START_REF] Pahlich | Protein arginine methylation. Cellular functions and methods of analysis[END_REF], this could explain the faster decline in valine and phenylalanine. An alternative hypothesis explaining the reduced levels of dimethylarginines and amino acids during the clamp might be an insulin-mediated suppression of their cellular release or stimulation of their uptake, which might reduce their circulating levels.

The current findings are in agreement with Eid et al [START_REF] Eid | Decreased levels of asymmetric dimethylarginine during acute hyperinsulinemia[END_REF] who demonstrated a 9% fall in plasma ADMA during a 90-minute euglycemic clamp in 20 male volunteers with borderline hypertension. They had also previously shown that, in vitro, insulin reverses the accumulation of ADMA in human umbilical vein and coronary artery endothelial cells incubated with TNFalfa by increasing DDAH activity [START_REF] Eid | Insulin and adiponectin inhibit the TNFα-induced ADMA accumulation in human endothelial cells. The role of DDAH[END_REF].

The data from the present study confirm a significant role for insulin in modulating plasma ADMA concentrations but do not help to explain the underlying therapeutic mechanism Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

responsible for the clinical benefit of intensive insulin therapy, in patients receiving intensive care [START_REF] Siroen | Modulation of asymmetric dimethylarginine in critically ill patients receiving intensive insulin treatment: a possible explanation of reduced morbidity and mortality?[END_REF]. It is seductive to conclude that reducing ADMA results in a favourable increase in NOS activity, but it may be that the fall in plasma ADMA simply reflects a beneficial effect of reducing protein catabolism or stimulating protein synthesis.

In non-diabetic subjects there is a significant correlation between plasma ADMA concentrations and insulin resistance [START_REF] Stuhlinger | Relationship between insulin resistance and an endogeneous nitric oxide synthase inhibitor[END_REF][START_REF] Mclaughlin | Plasma asymmetric dimethylarginine concentrations are elevated in obese insulin-resistant women and fall with weight loss[END_REF] and improvement in insulin sensitivity with rosiglitazone or weight loss results in a fall in ADMA [START_REF] Stuhlinger | Relationship between insulin resistance and an endogeneous nitric oxide synthase inhibitor[END_REF][START_REF] Mclaughlin | Plasma asymmetric dimethylarginine concentrations are elevated in obese insulin-resistant women and fall with weight loss[END_REF]. In obese and elderly subjects increased ADMA appears to be related to reduced insulin sensitivity [START_REF] Marliss | Elevations of plasma methylarginines in obesity and ageing are related to insulin sensitivity and rates of protein turnover[END_REF]. In contrast, we were unable to demonstrate any correlation between plasma ADMA and insulin sensitivity, assessed either by plasma insulin or insulin requirements to maintain euglycemia, during the overnight steady-state period. Similarly, the rate of decline of ADMA during the hyperinsulinemic clamp did not correlate with insulin sensitivity. These results are in agreement with data from women with gestational diabetes [START_REF] Mittermayer | Circulating concentrations of asymmetrical dimethyl-L-arginine are increased in women with previous gestational diabetes[END_REF] where increased ADMA levels were not found associated with insulin resistance. The discrepancies between studies might be related to differences in study populations, and/or in their degree of insulin resistance and associated insulin levels, or in the methods used to assess insulin sensitivity.

However, based on our finding of a relevant reduction in methylarginine levels following the acute infusion of high dose of insulin, it is likely that differences in plasma insulin levels in insulin resistant subjects might have the main role in influencing the rate of synthesis and/or catabolism of methylarginines. It could be argued that changes in insulin resistance related to different pubertal stages could have been another factor influencing the results of the present study. However, our study population included subjects all in late puberty (stage 4-5) and therefore the effect of differences in insulin resistance related to puberty can be ruled out .

SDMA has been proposed as a new marker of GFR [START_REF] Kielstein | Symmetric dimethylarginine (SDMA) an endogenous marker of renal function -a meta-analysis[END_REF][START_REF] Dalton | SDMA is an endogenous marker of glomerular filtration rate[END_REF] and, by competitive inhibition of arginine transport, as a cardiovascular risk factor [46]. In this study, plasma SDMA was independent of the large changes in glycemia, but did decline during the hyperinsulinemic euglycemic clamp. The implication is that the SDMA production rate will be dependent on protein turnover; an important consideration in any study using SDMA as an endogenous plasma GFR marker.

A limitation of the present study is related to the interpretation of the effect of hyperglycemia on dimethylarginines, since the best way to explore this effect would have been to perform a hyperglycemic clamp. Therefore, the apparent benign effect of the meal and subsequent changes in glucose levels need to be interpreted with caution. Furthermore, given that we did not specifically assess clearance of creatinine or ADMA, we cannot completely exclude the effect of variations in renal function during the all study on plasma ADMA levels. In conclusion, increases in insulin levels lead to rapid decline in ADMA levels, which parallel other markers of inhibition of protein catabolism, such as valine and phenylalanine. Acute changes in glucose levels do not appear to alter ADMA levels although further study is required. These 'acute effects' on ADMA need to be considered in the interpretation of longterm studies of association between ADMA and risk of complications in subjects with T1D. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Tables legend

Table 1 Characteristics of the study population Table 2 Overnight variable insulin infusion for euglycemia (18:00-04:00 h) Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

2 centrifuging for 3

 23 min at 21,800 g supernatants were transferred to a 96 deep well plate. For measurement of ADMA and SDMA, 5 µL of supernatant was automatically injected using an HTS PAL autosampler (CTC Analytics AG, Switzerland) into a 250 µL/min mobile phase stream of acetonitrile:water (50:50 v/v) with 0.025% (v/v) formic acid. Chromatography was performed on a Chirobiotic T 100 x 2.1mm column with a 2cm x 4.0mm guard column (Advanced Separation Technologies, Congleton, UK) and precursor/product ion pairs (m/z 203.1/46.2, 209.1/52.2 for ADMA and m/z 203.1/172.2, 209.1/175.1 for SDMA) were acquired in positive ion multiple reaction monitoring mode using a Sciex API4000 (Applied Biosystems, Warrington, UK). Assay standardisation was based on aqueous standards at 0.25, 1.0, and 5.0 µmol/l ADMA/SDMA, stored at -80 o C. Results were calculated using Analyst version 1.4.1. Pooled and spiked plasma samples were used for internal quality control. Intraassay variation (mean, CV%; n=10) was: ADMA 0.37 µmol/l, 2.1% and SDMA 0.44 µmol/l, 3.5%. Inter-assay variation (n=57) was: ADMA 0.39 µmol/l, 6.8%; 1.45 µmol/l, 5.5%; 4.20 µmol/l, 4.6% and SDMA 0.41 µmol/l, 3.2%; 1.88 µmol/l, 2.4%; 4.41 µmol/l, 3.3%. A second injection of the supernatant (1µl) was used for measurement of creatinine, arginine, valine, and phenylalanine. Solvent flow rate and chromatography were as above. Precursor/product ion pairs (m/z 114.2/44.2, 117.2/47.2 for creatinine, m/z 175.2/70.1, 179.2/71.1 for arginine, m/z 118.1/72.0, 126.1/80.0 for valine, and m/z 165.9/120.1, 170.9/125.1 for phenylalanine) were acquired in positive ion multiple reaction monitoring mode. Stock aqueous creatinine standard, 10 mmol/L, was prepared by dissolving creatinine (BDH (Chemicals) Ltd, Poole, UK) in 0.1M HCl and stored at 4 o C. The concentration of the stock standard has been confirmed using National Institute of Standards and Technology (NIST) standard reference material, creatinine 914a (Laboratory of the Government Chemist, Teddington, UK). Assay calibrators at 50, 250, and 1000 µmol/L were prepared from the stock standard by dilution with deionised water. Pooled and spiked quality control samples with assigned values determined using EU Community Bureau of Reference certified reference material sera (Report EUR 17115EN) CRM 573 (68.7 µmol/l), 574 (105.0 µmol/l), and 575 (404.1 µmol/l) (Laboratory of the Government Chemist, Teddington, UK) were included with each assay. Arginine stock aqueous standard, 10 mmol/l, was prepared from solid (Sigma-Aldrich Company Ltd, Poole, UK) and stored at -80 o C. Assay calibrators at, 10, 100, and 500 µmol/l were prepared from the stock standard by dilution with deionised water. A physiological fluid amino acid standard preparation (A9906, Sigma-Aldrich Company Ltd, Poole, UK) was used for standardisation, 25 and 250µmol/l, of the valine and phenylalanine assays. It provided independent standards at these concentrations for both creatinine and arginine. Pooled and spiked (creatinine and arginine) plasma samples were used for internal quality control. Interassay variation (n=56) was: creatinine 64.8 µmol/l, 3.5%; 184 µmol/l, 2.4%; 496 µmol/l, S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

  3 µmol/l (p=0.001), compared S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2008 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2008 Biochemical Society 46. Bode-Boger, S.M., Scalera, F., Kielstein, J.T. et al. (2006) Symmetrical dimethylarginine: a new combination parameter for renal function and the extent of coronary artery disease. J. Am. Soc. Nephrol. 17, 1128-1134 S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

Figure 1 Figure 2

 12 Figure 1 Plasma Glucose, Insulin, ADMA and SDMA during the overnight period (18:00-04:00h) (Error bars are 1 SEM)

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Figure 1

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  Figure 2

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Table 3 Overnight steady state period (04:00-08:00 am) Table 4 Hyperinsulinemic euglycemic clamp (08:00-12:00 am) Figures Legend

 3

Table 1 Characteristics of the study population Characteristics Mean (range)

 1

	Age (yr)	18.3 (13.2-24.4)
	Gender (M/F)	10/5
	Duration of diabetes (yr)	8.8 (0.8-12.0)
	HbA1c (%)	9.0 (6.4-13.6)
	Insulin dose (U/kg/day)	1.1 (0.7-1.6)
	BMI (kg/m 2 )	22.5 (18.4-25.3)
	Lean Body Mass (Kg)	56.8 (36. 6-71.0)
	Data are mean (range); BMI: body mass index; HbA1c: haemoglobin A1c

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

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Table 2 Overnight variable insulin infusion for euglycemia (18:00-04:00 h) TIME P values 18:00 h 20:15 h* 04:00 h 18:00 vs 20:15 20:15 vs 04:00 18:00 vs 04:00

 2

	ADMA (µmol/l)	0.446±0.073	0.475±0.073	0.479±0.058	<0.001	0.23	0.06
	SDMA (µmol/l)	0.411±0.056	0.408±0.062	0.401±0.052	0.80	0.06	0.32
	ADMA/SDMA ratio	1.08	1.11	1.17	0.09	0.90	0.06
		[0.83-1.40]	[1.0-1.5]	[0.91-1.58]
	Arginine (µmol/l)	36.6±10.8	47.9±13.5	52.1±12.3	<0.001	0.008	0.001
	Creatinine (µmol/l)	59.2±9.7	61.0±9.8	60.6±9.0	0.34	0.10	0.12
	Valine (µmol/l)	217±37	257±48	239±44	0.003	0.004	0.13
	Phenylalanine(µmol/l)	65.2±7.0	84.2±14.0	58.8±10.9	<0.001	<0.001	0.002
	Glucose (mmol/l)	11.0 [4.0-20.7]	13.6 [9.1-23.2]	5.2 [4.3-6.7]	0.18	<0.001	0.001
	Insulin (pmol/l)	99 [62-511]	346 [120-863]	73 [62-181]	<0.001	<0.001	0.05
	Data are mean±SD or median [range];
	ADMA: asymmetric dimethylarginine; SDMA: symmetric dimethylarginine
	*corresponding to the time when glucose peak was reached

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 3 Overnight steady state period (04:00-08:00 am)

 3

			TIME
		04:00 h	08:00 h	p
	ADMA (µmol/l)	0.479 ± 0.058	0.468 ± 0.056	0.26
	SDMA (µmol/l)	0.401 ± 0.052	0.414 ± 0.046	0.07
	ADMA/SDMA ratio	1.21 ± 0.19	1.14 ± 0.17	0.31
	Arginine (µmol/l)	52.1 ± 12.3	55.7 ± 13.7	0.27
	Creatinine (µmol/l)	60.6 ± 9.0	60.5 ± 9.2	0.89
	Valine (µmol/l)	239±44	213 ± 35	<0 001
	Phenylalanine (µmol/l)	58.8 ± 10.9	52.9 ± 9.8	0.001
	Glucose (mmol/l)	5.2±0.6	4.9±0.5	0.13
	Insulin (pmol/l)	82±32	115±16	0.02
	Data are mean±SD, ADMA: asymmetric dimethylarginine; SDMA: symmetric
	dimethylarginine

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079

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Table 4 Hyperinsulinemic euglycemic clamp (08:00-12:00 am) TIME 08:00 h 10:00 h 12:00 h p

 4

	ADMA (µmol/l)	0.468±0.056	0.436±0.065	0.364±0.050	<0.001
	SDMA (µmol/l)	0.414±0.046	0.402±0.047	0.373±0.040	<0.001
	ADMA/SDMA ratio	1.11 [0.92-1.49]	1.06 [1.02-1.22]	0.95 [0.77-1.31] <0.001
	Arginine (µmol/l/min)	55.7±13.7	55.1±12.7	42.9±10.7	<0.001
	Creatinine (µmol/l)	60.5±9.2	60.9±11.3	59.2±10.7	0.08
	Valine (µmol/l)	213±35	184±29	142±21	<0.001
	Phenylalanine (µmol/l)	52.9±9.8	46.9±9.4	37.4±8.4	<0.001
	Glucose (mmol/l)	4.9 [4.0-5.9]	4.9 [4.8-5.1]	4.8 [3.7-5.0]	0.19
	Insulin (pmol/l)	104 [62-296]	139 [83-674]	465 [228-574]	<0.001
	Data are mean±SD, ADMA: asymmetric dimethylarginine; SDMA: symmetric
	dimethylarginine

© The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

This study was supported in part by an ESPE Research Fellowship (to M.L.M), sponsored by Novo Nordisk A/S. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 22 May 2008 as manuscript CS20080079 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20080079 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © The Authors Journal compilation © 2008 Biochemical Society