Dr Hans-Jürgen J Brömme

Leoni Zühlke

Rolf-Edgar Silber

Andreas Simm

DCFH 2 interactions with hydroxyl radicals and other oxidants -influence of organic solvents

Keywords: DCFH 2, hydroxyl radicals, ethanol, dimethyl sulfoxide, hydrogen peroxide, heme-proteins

A C C E P T E D

M A N U S C R I P T 2

Abstract

The oxidation of 2',7'-dichlorofluorescin (DCFH 2 ) is widely used as a measure to detect the generation of reactive oxygen species (ROS) and to analyze oxidative stress. Other factors beside commonly known radicals may influence the results of such measurements. Therefore, the effects of H 2 O 2 , KMnO 4, decomposition products of AAPH, ethanol and DMSO, antioxidants like ascorbic acid, different ferrous ion chelates, and heme-containing proteins like cytochrome c, myoglobin, hemoglobin, and horseradish-peroxidase were comparatively analyzed with respect to their impact on DCFH 2 oxidation. The study evaluates the effects of various oxidants with different oxidative potentials regarding their ability to induce DCFfluorescence. Furthermore, we analyzed the inhibitory effect of organic solvents like ethanol or DMSO on the oxidation of DCFH 2 by hydroxyl radicals. The results of our study indicate that the potential of an oxidant does not always correlate with its efficiency to generate DCFfluorescence.

Introduction

Free radical formation and oxidative stress play an important role in the etiology of several degenerative diseases [START_REF] Halliwell | Oxygen-derived species: their relation to human disease and environmental stress[END_REF]. Harman proposed 50 years ago that aging is the result of the accumulation of oxidative damage from radicals generated endogenously in cells during normal metabolism (Harman, 1956). Trying to find a partner for its lone electron, the free radical captures an electron from another molecule, which in turn becomes unstable and reacts with further molecules [START_REF] Simm | Reactive oxygen species (ROS) and aging: Do we need them -can we measure them -should we block them?[END_REF]. Harmful molecules can arise which damage proteins, membranes, and nucleic acids, particularly DNA. There is a linkage between the age-dependent accumulation of oxidized enzymes and the loss of physiological function [START_REF] Stadtman | Protein modification in aging[END_REF]. By using models like transgenic mice and fruit-flies it has been shown that modification of the antioxidant defence mechanisms like the superoxide dismutase (SOD) can influence the ageing process and thereby modify the life span [START_REF] Landis | Superoxide dismutase evolution and life span regulation[END_REF]. In contrast, radicals are an important physiological integral part of signaling pathways as well as of defense mechanisms of neutrophiles and macrophages [START_REF] Epling | Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method[END_REF][START_REF] Simm | Reactive oxygen species (ROS) and aging: Do we need them -can we measure them -should we block them?[END_REF]. Therefore, in many laboratories, including those, which investigate the impact of radical species on processes involved in the pathogenesis of degenerative diseases and in aging, the measurement of the formation of reactive oxygen species (ROS) is of increasing importance. The methods used should be easy to perform and valid regarding the determined reactive species. A frequently used method is the oxidation of the non-fluorescent probe 2´,7´-dichlorodihydrofluorescein (DCFH 2 ). DCFH 2 in its acetylated form (DCFH 2 -DA) can easily pass lipid membranes and after deacetylation by cellular esterases can be oxidized to form the fluorochrome 2´,7´dichlorofluorescein (DCF), which upon excitation at 480 nm emits light at 520 nm. This method has been widely used in flow cytometry, fluorescence microplate assays, or fluorescence microscopy [START_REF] Silveira | Formation of hydrogen peroxide in rat skeletal muscle cells during contraction[END_REF][START_REF] Tada | Nitric oxide and reactive oxygen species do not elicit hypersensitive cell death but induce apoptosis in the adjecent cells during the defence responce of oat[END_REF][START_REF] Watanabe | In vivo fluorometric measurement of cerebral oxidative stress using 2´-7´-dichlorofluororescin (DCF)[END_REF]). Originally, it has been assumed that DCF-fluorescence appears as a consequence of the formation of superoxide anion radicals or hydrogen peroxide

A C C E P T E D M A N U S C R I P T 4
and, therefore, has been regarded as a measure for the generation of this reactive oxygen species [START_REF] Al-Mehdi | Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+[END_REF][START_REF] Hempel | Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123[END_REF][START_REF] Kurose | Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication[END_REF]. Recently it became obvious that oxidation of different fluorescent probes (including DCFH 2 ) is less a result of particular oxidizing species but rather of a generalized oxidative stress [START_REF] Halliwell | Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?[END_REF]. The present investigation analyzes the influence as well as the individual impact of different compounds on DCFH 2 oxidation and DCF fluorescence, examines the influence of ethanol and DMSO as commonly used solvents for DCFH 2 regarding their effects on generation of fluorescence induced by •OH, and interprets data concerning their influence on cellular systems. The fluorescence in the sample was monitored 2.5 min after starting DCFH 2 -oxidation in a fully automated microplate based multi-detection reader FLUOstar OPTIMA from BMG Labtech GmbH (Offenburg, Germany) using identical measuring conditions (excitation: 485 nm, emission: 520 nm, gain 1500, 25 °C).

Materials and methods

Material

Sample composition for electron spin resonance (ESR)-spectroscopy

The blank (control) consisted of 80 µL of water, 5 µL of DMPO (1 mol/L stock), 10 µL of

Statistical analysis

Data are expressed as mean ± standard deviation (SD) of at least 5 independent experiments.

For comparison of means the Student´s t-test was applied. P-values less than 0.05 were considered significant.

Results

Several chemical oxidants, like e.g. alkylperoxyl radicals (decomposition products of AAPH formed in the presence of oxygen) [START_REF] Niki | Free radical initiators as source of water-or lipid-soluble peroxyl radicals[END_REF] or KMnO 4 are able to induce a concentrationdependent oxidation of DCFH 2 . Hydrogen peroxide, which is often used as a positive control in cell culture experiments, oxidizes DCFH 2 in the absence of additional factors only to a very small degree, if at all (Fig. 1A-C ). However, if ferrous ion chelates of NTA, EDTA or citrate are present, DCFH 2 will be rapidly oxidized by H 2 O 2 (Fig. 2A). In the absence of Eq. I:

Fe 2+ + H 2 O 2  Fe 3+ + •OH + OH -
Eq. II:

•OH + DMPO  •DMPO-OH
Eq. III: Fe 3+ + ascorbate  Fe 2+ + ascorbate radical [START_REF] Udenfriend | Ascorbic acid in aromatic hydroxylation. I. A model system for aromatic hydroxylation[END_REF] Heme-containing proteins like cytochrome c, myoglobin, hemoglobin, or peroxidases produce much more DCF from DCFH 2 than the •OH-triggered oxidation of DCFH 2 . The higher the concentration of the heme-containing protein the more intense will be the fluorescence (Fig. 3A-D). Without H 2 O 2 no DCFH 2 -oxidation is observed (Fig. 3A).

Discussion

Hydroxyl radicals (•OH) belong to the most aggressive radicals known in biology [START_REF] Buettner | Spin trapping: ESR parameters of spin adducts[END_REF]Halliwell and Gutteridge 1999;[START_REF] Mason | Detection of oxygenderived radicals in biological systems using electron spin resonance[END_REF]. Their oxidizing potential is higher than that of any other known cellular oxidant [START_REF] Buettner | The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate[END_REF]). Nevertheless, the ability of

•OH to oxidize DCFH 2 appears to be only modest when compared with the oxidizing potential of KMnO 4 , AAPH derived alkylperoxyl radicals, and the oxidizing intermediates originated from heme-containing proteins.

How can this discrepancy be explained? The higher the oxidizing potential of a compound, the higher will be its chance to react with any other electron donating compound in its immediate vicinity. Although the composition of the reaction mixture is of low complexity, nevertheless, beside DCFH 2 the sample contains some additional targets for •OH. The most important of these supplementary targets are the organic solvents (ethanol and DMSO) in which DCFH 2 is dissolved. From both solvents it is known that they are excellent scavengers of •OH [START_REF] Cederbaum | The effect of dimethylsulfoxide and other hydroxyl radical scavengers on the oxidation of ethanol by rat liver microsomes[END_REF][START_REF] Heikkila | The prevention of alloxan-induced diabetes in mice by dimethyl sulfoxide[END_REF][START_REF] Heikkila | Prevention of alloxan-induced diabetes by ethanol administration[END_REF]. This means that if such

A C C E P T E D M A N U S C R I P T

8 additional targets are present, they compete with DCFH 2 for the •OH. The probability from which target a hydrogen atom will be abstracted depends on the local concentrations of all potential targets. The concentration ratio of ethanol, DMSO and DCFH 2 in the sample amounts to 15400 : 1400 : 8 µmol/L. Therefore, DCFH 2 should not be the favored target for

•OH. Under the conditions applied, a maximum of 25 µmol/L •OH could be generated.

However, the trapping efficiency (the chemical yield of the diamagnetic spin trap converted to the paramagnetic adduct) is normally less than 100 %. From DMPO it is known that its trapping efficiency amounts to 33 % [START_REF] Schwarz | A new method to determine the generation of hydroxyl radicals in illuminated TiO2 suspensions[END_REF]. A corresponding trapping efficiency for DCFH 2 is not known. But it can be assumed that most of DCFH 2 present in the sample should be oxidized by this strong oxidant. Assuming an approximately similar electron donating potential for these three competing compounds, it can be expected that both organic solvents must be the preferred targets for •OH. This is indeed substantiated by the ESR experiments. As a result, the intensity of the •DMPO-OH adduct signal formed in the presence of DMSO or ethanol is remarkably lower than the signal intensity in the absence of organic solvents (see Fig. 2B). Regarding the preferred interaction of ethanol and DMSO with

•OH the low efficiency of •OH to oxidize DCFH 2 can therefore be understood. The low efficiency of •OH to oxidize DCFH 2 was also reported by others [START_REF] Lebel | Evaluation of the probe 2',7'dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress[END_REF].

However, until now it remains unclear whether the carbon-centered radicals derived from the

•OH driven oxidation of organic solvents are able to participate also in the oxidation of DCFH 2 .

Under certain conditions the presence of ascorbate can intensify the formation of •OH from H 2 O 2 and iron chelates [START_REF] Udenfriend | Ascorbic acid in aromatic hydroxylation. I. A model system for aromatic hydroxylation[END_REF]. Under these conditions ascorbate probably acts as a pro-oxidant by reducing ferric ions back into their catalytically active state (redoxcycling of iron ions at the expense of ascorbate, see equations I-III).

On the other hand, oxidants less potent than •OH, like e.g. KMnO 4 or alkylperoxyl radicals, seem to be nearly ineffective in oxidizing DMSO or ethanol. Therefore they oxidize mainly DCFH 2 and produce significantly more DCF-fluorescence.

Heme-proteins, like cytochrome c [START_REF] Burkitt | Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis[END_REF][START_REF] Burkitt | Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis[END_REF][START_REF] Lawrence | Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome c/H2O2. Implications for oxidative stress during apoptosis[END_REF], myoglobin or hemoglobin, can act as pseudoperoxidases. This heme-containing proteins, in general, lack substrate specificity. Hydrogen peroxide, organic peroxides, and molecular oxygen all can serve as oxidizing agents, while the list of reducing agents is even longer. The oxidizing entity of pseudoperoxidases appears to be compound I, a transiently formed heme intermediate [START_REF] Tarpey | Methods of detection of vascular reactive species: nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite[END_REF]. It is believed that compound I consists of a ferryl (Fe IV ) species and a porphyrin radical state of heme which abstracts electrons from the reductant, thereby regenerating the catalytic activity of the pseudoperoxidase [START_REF] Cartoni | Oxidative degradation of cardiotoxic anticancer anthracyclines to phthalic acids. Novel function or ferrylmyoglobin[END_REF][START_REF] Goldman | Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron[END_REF][START_REF] Kawano | Spectroscopic evidence in support of horseradish peroxidase compound II-catalyzed oxidation of salicylic acid but not of phenylethylamine[END_REF]. Under the in vitro conditions applied, the only available reductant seems to be DCFH 2 . Therefore, DCFH 2 is oxidized continuously by the pseudoperoxidases. The higher the amount of pseudoperoxidases, the more DCFH 2 is oxidized per unit of time. Thus DCFH 2 -oxidation is limited mainly by the availability of H 2 O 2 . Since in our batch natural substrates (cellular reductants) for peroxidases were omitted, horse radish peroxidase (hr-POD) utilizes only DCFH 2 as an artificial reductant. It might be expected that in the presence of e.g. GSH or other reductive antioxidants, the rate of oxidation of DCFH 2 would decline significantly as has been shown recently [START_REF] Lawrence | Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome c/H2O2. Implications for oxidative stress during apoptosis[END_REF].

Which scenario can be expected in cells? Which oxidants could be able to oxidize DCFH 2 in a cellular environment? Superoxide anion radicals (•O 2 -) and hydrogen peroxide are continuously produced by mitochondria, by certain enzymes, as well as by some autoxidation reactions. The steady state concentration of both ROS normally is kept low, since superoxide dismutases, catalase, and several peroxidases minimize their availability. Nevertheless, in the presence of "free" transition metals, FENTON chemistry always produces a certain amount of [START_REF] Burkitt | Cytochrome C is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis[END_REF]. On the other hand, •OH possesses this potential, but the probability of oxidizing DCFH 2 should be rather low, since many alternative cellular targets compete with DCFH 2 for this highly reactive oxidant. Scheme 1 summarizes the different effectors for DCFH 2 oxidation.

A C C E P T E D M A N U S C R I P T 10 •OH from H 2 O 2 ,
The primary derivatives of an attack of •OH on cellular compounds will be mostly again radicals. Some of them, like e.g. the alkylperoxyl radicals formed during lipid peroxidation, are obviously able to oxidize DCFH 2 (see Fig. 1B). Other radicals, like the ascorbate-radical or carbon-centered radicals, will probably miss this ability.

Interestingly, this scavenging reaction of ethanol against hydroxyl radicals may be a potential reason for some positive effects of low amounts of ethanol on the life span seen in humans.

For example, it could be demonstrated that moderate alcohol consumption can clearly reduce the risk of cardiovascular diseases in populations throughout the world [START_REF] Rimm | Alcohol in the Mediterranean diet[END_REF]. The current recommendation from the British Heart Foundation is a maximum consumption of 30 g ethanol per day [START_REF] Preedy | Ethanol induced cardiovascular disease[END_REF]. Epidemiological studies have shown the protective effect of modest alcohol consumption on the incidence of cardiovascular diseases which account for the majority of death in Western world [START_REF] Redmond | Potential mechanisms for cardiovascular protective effect of ethanol[END_REF][START_REF] Tolstrup | Prospective study of alcohol drinking patterns and coronary heart disease in women and men[END_REF]. We could recently demonstrate that at a final concentration of about 0.1 promille (17 mM), ethanol effectively prevents degradation or modification of chemical compounds by hydroxyl radicals [START_REF] Brömme | Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction with melatonin and with other hydroxyl radical scavengers[END_REF][START_REF] Brömme | Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction with melatonin and with other hydroxyl radical scavengers[END_REF].

Moreover, when ethanol was dissolved in the perfusion buffer of isolated rat hearts to a final concentration of 21.7 mM (which corresponds to little more than 0.1 promille) a significant reduction of cell injury was observed after reoxygenation [START_REF] Ashraf | Beneficial effects of low doses of ethanol on reoxygenation injury following anoxia in rat hearts[END_REF].

On the other hand, when ethanol was administered in much higher quantities many harmful effects have been reported by various authors [START_REF] Ribiere | Effects of chronic ethanol administration on free radical defence in rat myocardium[END_REF][START_REF] Wieland | Oxidation of mitochondrial proteins and DNA following administration of ethanol[END_REF][START_REF] Mansouri | An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice[END_REF]. Above 80 g ethanol per day undoubtedly alcohol has ageaccelerating and life-shortening properties [START_REF] Laufs | Neues vom Alkohol-Optimum in der Prophylaxe von Herz-Kreislauf-Erkrankungen[END_REF][START_REF] Renaud | Alcohol and mortality in middle-aged men from eastern France[END_REF].

In summary, any transient decrease in the concentration of antioxidants (reductants) as well as any simultaneous increase in the formation of ROS as well as some ROS derived organic radicals will increase DCFH 2 -oxidation and thus fluorescence. On the other hand, hydroxyl radicals preferentially oxidize the organic solvents in cell-free batches whereas in cells mainly compounds in their immediate vicinity widely outcompete DCFH 2 . From experiments using DCFH 2 , one can therefore not conclude that increasing DCF fluorescence is indeed a proof for an increase of a particular radical formation. In ageing research, it will also be a problem to compare the oxidative stress in young and old cells due to changing cellular amounts of

  H 2 O 2 (20 mmol/L) and 5 µL of Fe 2+ (0.5 mmol/L). When different organic solvents were tested the composition of the mixture was: 67.5 µL of buffer, 20 µL of organic solvent (DMSO, Ethanol, DMF), 2.5 µL of H 2 O 2 , 5 µL of Fe 2+ , and 5 µL of DMPO. In the presence of organic solvents the mixture consisted of 60 µL of buffer, 20 µL of ethanol or DMSO, 5 µL of DMPO, 10 µL of H 2 O 2 and 5 µL of Fe 2+ . 50 µL of each mixture were immediately placed into disposable microtubes Brand GmbH (Wertheim, Germany) and sealed carefully with plasticine. The tubes were then placed into the cavity of an ESR-spectrometer Miniscope MS 100 Magnettech GmbH (Berlin, Germany).

  A C C E P T E DM A N U S C R I P T 6 Spectrometer settings were: modulation amplitude: 1 G, field scan: 100 G, scan time: 30 sec, number of repeats: 5, B 0 -field: 3365 G, power attenuation: 12 dB.

  H 2 O 2, iron chelates oxidize DCFH 2 only marginally (not shown). The oxidizing species in this type of experiments appear to be hydroxyl radicals (•OH) which are formed by the FENTON reaction according to equation I. ESR spectroscopy is the only method that allows a direct measurement of radicals. Due to the short half-life of oxygen derived radicals their monitoring needs the presence of a spin trap like DMPO. A diamagnetic spin trap can be transformed by a given radical into a paramagnetic derivative or adduct, which can be characterized by its individual ESR-spectrum. An ESR-spectrum corresponding to a •DMPO-OH adduct indicating the formation of hydroxyl radicals can be seen in Figure 2B, trace b. Without H 2 O 2 no •OH are generated und no •DMPO-OH adducts are formed (trace a). Ethanol as well as DMSO prevent greatly the formation of such adducts (trace d, e). But, if the spin trap (DMPO) is added after •OH has already interacted with ethanol and DMSO, the formed carbon-centered radicals produce mixed adducts with DMPO (trace c). Figure 2C A C C E P T E D M A N U S C R I P T 7 shows the influence of ascorbate on DCFH 2 oxidation induced by •OH. Here ascorbate acts predominantly as a pro-oxidant. According to equation III, ascorbate reduces the produced ferric ions back into their catalytically active ferrous state. The surplus of the formed •OH significantly increases the oxidation of DCFH 2 as seen by the increased fluorescence in the presence of ascorbate.

Figure 3 Scheme 1 :E

 31 Figure 3 Influence of different heme-containing proteins on DCF-fluorescence A.) Sample composition: 150 µL of buffer, 40 µL of DCFH 2 (40 µM stock), 5 µL of cytochrome c (5 or 10 µM stock) ± H 2 O 2 (20 mM stock). B.) Sample composition: 150 µL of buffer, 40 µL of DCFH 2 (40 µM stock), 5 µL of hr-POD (0.1, 0.25, 0.5 µM stock), 5 µL of H 2 O 2 (20 mM stock). C.) Sample composition: 150 µL of buffer, 40 µL of DCFH 2 (40 µM stock), 5 µL of H 2 O 2 (20 mM stock), 5 µL of myoglobin (1 to 10 µM stock). D.) Sample composition: 150 µL of buffer, 40 µL of DCFH 2 (40 µM stock), 5 µL of H 2 O 2 (20 mM stock), 5 µL of hemoglobin (1 to 5 µM stock). For further details see Fig. 1A.

  To 145 µL of phosphate buffer according to Soerensen (27.4 parts KH 2 PO 4 25 mmol/L and 72.6 parts Na 2 HPO 4 x 2 H 2 O, 25 mmol/L, pH 7.2) 40 µL of DCFH 2 (40 µmol/L stock), 5 µL of H 2 O 2 (20 mmol/L stock) and 10 µL of an oxidizing compound were added into 96 Well Plates, Greiner, Omega Scientific (Venture, CA, USA). In the case of chemical oxidants, like KMnO

	neutralized with 1000 µL of phosphate buffer (25 mmol/L stock). After neutralization, the
	final concentration of DCFH 2 (total 1250 µL) amounted to 40 µmol/L. The concentration of
	DMSO and ethanol in the mixture was 56 and 616 mmol/L respectively.
	2.3. Sample composition for fluorescence measurements
	2´,7´-dichlorodihydrofluorescein diacetate (DCFH 2 -DA), ascorbic acid (AA), cytochrome c
	(Cyt c) from equine heart, dimethylformamide (DMF), dimethylsulfoxide (DMSO),
	ethylenediaminetetraacetic acid (EDTA), ferrous sulphate heptahydrate (FeSO 4 x 7 H 2 O),
	ferric chloride hexahydrate (FeCl 3 x 6 H 2 O), hydrogen peroxide (H 2 O 2 ), myoglobin (Mb)
	from equine heart, nitrilotriacetic acid (NTA), and potassium permanganate (KMnO 4 ) were
	purchased from Sigma (Taufkirchen, Germany), 5,5-Dimetyl-1-pyrroline-N-oxide (DMPO)
	was obtained from Alexis (Grünberg, Germany), Citric acid and Water (HPLC grade) were
	from Merck (Darmstadt, Germany), hemoglobin (Hb), and peroxidase from horseradish (hr-
	POD) were purchased from ICN Biomedicals GmbH (Eschwege, Germany). 2´,2´-azobis-2-
	methyl-propanimidamide, dihydrochloride (AAPH) was obtained from Polysciences Inc.
	(Warrington, PA, USA). All other chemicals were obtained from Fluka (Taufkirchen,
	Germany).
	2.2. In vitro deacetylation of DCFH 2 -DA
	5 µL of DCFH2-DA (10 mmol/L dissolved in DMSO or DMF) and 45 µL of ethanol (96 %
	v/v) were hydrolyzed in the presence of 200 µL of NaOH (10 mmol/L stock) and thereafter

4 or AAPH, hydrogen peroxide was not present in the sample. When hemoproteins were used the composition of the sample was slightly different: To 150 µL of buffer, 40 µL DCFH 2 , 5 µL of H 2 O 2 and 5 µL of either Cyt c, Mb, Hb or POD were pipetted. The control consisted of 160 µL of buffer and 40 µL of neutralized DCFH 2 .

  and •O 2 -help to re-reduce Fe(III) to its catalytically active form (Fe(II)). But neither •O 2 -nor H 2 O 2 seem to have a potential high enough to induce DCFH 2 -derived fluorescence. Both need additional factors in order to oxidize DCFH 2

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Glycation associated skin autofluorescence and skin elasticity are related to chronological age and body mass index of healthy subjects

published or not. The documents may come     Glycation associated skin autofluorescence and skin elasticity are related to chronological age and body mass index of healthy subjects

A C C E P T E D

M A N U S C R I P T Since the formation of AGEs is a slow process, the body's long-lived proteins such as collagen are mostly susceptible to AGE accumulation. AGEs have been known to accumulate in various tissues as a function of chronological age [START_REF] Hipkiss | Accumulation of altered proteins and ageing: causes and effects[END_REF]. This build up of glycated modifications happens at an even much higher rate in several age-related diseases like diabetes (Dyer et al., 1993), vascular (Soldatos andCooper, 2006) and neurodegenerative (Sasaki et al., 1998) diseases and osteoarthritis (Senolt et al., 2005).

Skin is a tissue in which the formation and accumulation of AGEs has been extensively studied and the presence of glycation products in the skin has been proposed as a marker and predictor of the progression of certain diseases [START_REF] Monnier | Glycation products as markers and predictors of the progression of diabetic complications[END_REF]. The initial method for quantifying AGEs was based on the measurement of collagen-linked fluorescence in biopsies [START_REF] Odetti | Age-related increase of collagen fluorescence in human subcutaneous tissue[END_REF]. The age related trends observed with this technique were later confirmed using hyphenated analytical tools that enabled the analysis of specific AGEs like N  -(carboxymethyl)lysine and pentosidine [START_REF] Dyer | Accumulation of Maillard reaction products in skin collagen in diabetes and aging[END_REF]. Recently skin autofluorescence has been proposed as yet another non-invasive clinical tool to study age related phenomena in human skin [START_REF] Kollias | Endogenous skin fluorescence includes bands that may serve as quantitative markers of aging and photoaging[END_REF]. Skin autofluorescence, collected at the excitation and emission wavelength of 370 and 440nm respectively is assumed to be specific for (collagen linked) AGEs. Skin autofluorescence has been shown to be correlated with the presence of specific AGEs (Meerwaldt et al., 2005b) and was associated to chronological age and diabetes duration [START_REF] Meerwaldt | Simple non-invasive assessment of advanced glycation endproduct accumulation[END_REF].

Although the analyses of AGEs in skin has been very often used as a marker of AGE accumulation, only recently the impact of the glycation process on the structure and functioning of the skin itself has been explored. In the current study we have used in

A C C E P T E D M A N U S C R I P T

1

Subjects

Healthy female volunteers, with Fitzpatrick skin type II or III, who were in good general health, free of any dermatologic disorders, and who gave their informed, written consent, were allowed to enter the studies.

Clinical measurements

In vivo skin autofluorescence measurements were collected with an LS 50B fluorescence spectrometer (PerkinElmer, Waltham, MA, USA) equipped with a fibre optic. The excitation and emission wavelengths were set to 370 and 440 nm respectively and data were collected on the ventral forearm of the panellists. On the same site, skin colour was measured with a chromameter (Minolta, Osaka, Japan) and expressed as Individual Typology Angle (ITA°). Pigmented skin is characterised by a low ITA-value. Height was measured and body weight recorded using a Terraillon TFX70 scale (Terraillon, Paris, France). Body mass index (BMI) was calculated by dividing the weight of the panellist in kilograms by the square of the height in meters.

A BMI of 20 to 25 indicate normal weight; a number from 25 to 30 indicate the person is overweight and a number above 30 suggests the person is obese.

Skin elasticity was measured on the outer thigh of the panellists with a Torsional ballistometer (Dia-Stron Ltd., Andover, UK). Elasticity of the skin was expressed as the rate of energy damping A high -value is indicative for a reduced elasticity.

Statistical analysis

Statistical calculations were performed using Statistica 6.0 (Statsoft, Tulsa, OK, USA). Data were analysed by single and multiple linear regression analysis. A probability level of 0.05 was chosen as the point at which differences were considered significant.

materials and methods

A C C E P T E D M A N U S C R I P T

1

AGE-associated fluorescence as a function of chronological age and BMI

In the first study (study A) skin autofluorescence data were collected on the ventral forearm of 448 female Caucasian panellists. The group characteristics are summarised in Table 1. Besides skin autofluorescence, the skin colour, expressed as Individual Typology Angle (ITA), was measured on the same site. There existed a strong inverse correlation between the skin colour and the fluorescence intensity (n=448; r=0.76, p<10 -17 ). The dataset was broken up in two groups, depending on the chronological age of the panellist viz. below or above the age of 40. For each of both datasets, multiple linear regression analysis was used to investigate the independent effects of skin colour, chronological age and BMI on the skin autofluorescence. Multicollinearity statistics revealed that tolerance values were typically above 0.9 hence the individual parameters were independent. Pareto charts are shown in Fig. 1 for both the younger panellists (top panel) and for the group of the older panellists (bottom panel).

The statistical outcome is summarised in Table 2. For both age groups the bar for the skin colour crossed the dotted line, which marks the critical value for statistical significance (Fig. 1). The fitted parameters for each variable are listed in Table 2 and this indicates that an increase in the ITA value, and thus a less pigmented skin, is associated to a higher autofluorescence value. Out of the three variables studied, ITA was clearly the most dominant one. For the young-age group, both age and BMI were positively associated to the in vivo AGE-associated fluorescence signal (Fig. 1 and Table 2). For the older panellists the dependence of the fluorescence on chronological age was maintained, but there was no statistically significant association with the BMI. This suggests that only for younger panellists, both BMI and chronological age were recognised as being factors positively associated to the autofluorescence signal, while in the old age group the effect of age on the fluorescence was much stronger and became dominant over the BMI effect.

Skin elasticity as a function of chronological age and BMI

Eighty-four female panellists were enrolled in the clinical study (study B) and the group characteristics are summarised in Table 1. Skin elasticity was measured with a torsional ballistometer and expressed as the rate of energy damping . Data were collected on the outer thigh, a body site that is prone to fat accumulation in women.

The panellists were subdivided in two age groups and the number of panellists results

A C C E P T E D M A N U S C R I P T

2 assigned to the young age (< 40 years) and older age (≥ 40 years) group was 30 and 54 respectively. Statistical evaluation of the data proceeded in a similar fashion as for the first study, except that skin colour was not considered as being a relevant parameter for skin elasticity. Data are summarised in Fig. 2 and in Table 3. For the young age group, a decrease in skin elasticity was mainly associated with high BMI values but there was no significant correlation with panellist age. For the older age group, the opposite was observed, and the skin elasticity was largely affected by the age of the panellist but not by the BMI.

A C C E P T E D M A N U S C R I P T

1 Several methods exist for the measurement of AGEs in skin samples. Some of these methods require the sampling of biopsies and are invasive in nature. Recently skin autofluorescence has been proposed as a non-invasive tool to study AGEs in human skin [START_REF] Meerwaldt | Simple non-invasive assessment of advanced glycation endproduct accumulation[END_REF].

A clinical study was conducted in which the glycation associated fluorescence and skin colour were measured on the ventral forearm of 448 female healthy Caucasian panellists. Their Fitzpatrick skin type was II to III and they had similar skin tone with a median ITA value of 48.0 (Table 1). Nevertheless there existed a very strong inverse correlation between skin colour and fluorescence (p<10 -17 ). This agrees with observations already described in the literature [START_REF] Na | Autofluorescence of human skin is age-related after correction for skin pigmentation and redness[END_REF]. Pigmented skin most probably contains compounds that absorb or scatter the fluorescent light and as a result the fluorescence intensity decreases. Various methods have been proposed to anticipate for this strong dependence of the fluorescence intensity on skin colour [START_REF] Sandby-Moller | Influence of epidermal thickness, pigmentation and redness on skin autofluorescence[END_REF]Meerwaldt et al., 2005a;Stamatas et al., 2006). We have used multiple linear regression analysis tools to study the skin autofluorescence as a function of skin colour, chronological age and BMI. The latter, which is a measure for the panellist's overweight was considered here since an increase in overweight and obesity prevalence is evident in the Western societies over the last decades [START_REF] Hedley | Prevalence of overweight and obesity among US children, adolescents, and adults, 1999-2002[END_REF]Tzotzas and Krassas, 2004). In this study the median BMI of the panellists was 26 ± 6. 2 and figure 1). For the older age panellists the age effect was maintained but the effect of BMI on the glycation associated fluorescence was not conserved. These data suggest that skin AGEs accumulated as a function of chronological age, which agrees with many other studies (Sell et al. 1996;[START_REF] Jeanmaire | Glycation during human dermal intrinsic and actinic ageing: an in vivo and in vitro model study[END_REF][START_REF] Dyer | The Maillard reaction in vivo[END_REF]. BMI however only seemed to be associated with the glycation associated fluorescence in the young age group. This might be explained by an increase in the rate of accumulation of skin AGEs at higher chronological age. Within the older age group, the effect of age on the fluorescence became dominant over BMI.

A second clinical study was conducted in which skin elasticity of 84 female panellists was measured. Mechanical properties of the skin and elasticity in particular can be evaluated with a number of instruments based on torsion or extension methods. In this study a torsional ballistometer was used. This instrument records the bouncing profile of a small hammer that is dropped on the skin surface with a constant force. Earlier it has been reported that skin elasticity measured with the ballistometer decreased in an old (50-67 years) versus a young population (18-28 years) [START_REF] Agarwal | Variability in the physical properties of the stratum corneum: influences of chronological age and season[END_REF]. In this study an additional parameter viz. BMI was considered. The impact of obesity on skin physiology has been recognised before (Yosipovitch et al., 2007), but the evaluation of normal to slightly overweight panellists forms the focus of this study.

From Table 1 it follows that the age and BMI distribution was very similar to the first study. Statistical treatment of skin elasticity data was identical to study A, except that the skin colour, which was not associated to skin elasticity, was omitted as an independent variable in these calculations. The dependence of the skin elasticity, expressed as the -value, on chronological age and BMI was different for both agegroups. For the young age group, skin elasticity was negatively associated to BMI but not to chronological age. The opposite was observed in the older age panellists (Fig. 2 and Table 3).

There was similarity in the way that both glycation associated autofluorescence and play an important role in the stiffening of plantar skin in diabetes [START_REF] Hashmi | Plantar skin in type II diabetes: an investigation of protein glycation and biomechanical properties of plantar epidermis[END_REF]. This also agrees with data showing that in vitro glycated skin samples are characterised by a loss of biomechanical properties [START_REF] Reihsner | Alterations of biochemical and two-dimensional biomechanical properties of human skin in diabetes mellitus as compared to effects of in vitro non-enzymatic glycation[END_REF].

In summary measurement of in vivo skin autofluorescence is proposed as a noninvasive clinical tool that enables the measurement of skin AGE accumulation in healthy panellists. This method was used to evaluate skin AGE accumulation as a function of chronological age and BMI of the panellist. Furthermore skin elasticity data also showed a dependence on chronological age and BMI, leading to the hypothesis that collagen glycation or skin AGE accumulation in general is one of the parameters that contributes to the loss of skin elasticity.

A C C E P T E D M A N U S C R I P T

  of glycation, reducing sugars react non-enzymatically with target molecules like proteins, lipids or DNA. During the first stages of this process, early glycation products are reversibly formed. Side products generated in the intermediate steps may be oxidising agents and/or reactive carbonyl compounds like (methyl)glyoxal and glycolaldehyde. Next these early glycation products react further to give rise to a whole set of modifications collectively termed advanced glycation endproducts (AGEs), from which some of them are characterised by intra-and intermolecular cross-linking and typical fluorescence bands (Ulrich and Cerami, 2001).

  measurements to assess skin AGE accumulation in healthy volunteers as a function of chronological age and body mass index (BMI). Data are presented to support the link between AGE accumulation and the in vivo loss of skin elasticity.

  3 and the majority of the panellists had a BMI between 20 and 30, indicating that this study was primarily focussed towards normal to overweight people. The age of the panellists ranged from 19 up to 70 years, with about hundred panellists for each decade of age. Formerly it was shown that pentosidine in skin collagen increased in an age dependent manner(Sell et al. 1996) but at relatively young age, AGE levels were low and remained fairly constant. Only above the age of 40 the rate at which these AGEs accumulated strongly increased. Immunostaining experiments of skin samples showed similar trends[START_REF] Jeanmaire | Glycation during human dermal intrinsic and actinic ageing: an in vivo and in vitro model study[END_REF]. The same observation was made in human cartilage collagen where during the first decades of life, levels of AGEs remained low but at higher age the concentration of the AGEs increased up to 30 fold(Verzijl et al., 2000). This age dependence of AGE accumulation urged us to break up the dataset in two, based on the age of the panellist. Statistical calculations were then performed independently for the young age group (<40 years) and for the older age group (≥ 40 years). Within a population of normal to overweight, young (< 40 years) and healthy panellists, both chronological discussion A C C E P T E D M A N U S C R I P T 2 age and BMI contributed in a statistically significant way to the increase in the glycation associated fluorescence (Table

Fig. 1 Fig. 2

 12 Fig. 1 Pareto charts of the multiple linear regression analyses on the association between in vivo skin fluorescence and skin colour (ITA), chronological age and BMI. Results for the young age and old age group are shown in the top and bottom panel respectively.

1

  Glycation associated skin autofluorescence and skin elasticity are related to chronological age and body mass index of healthy subjects H. Corstjens 1* , D. Dicanio 2 , N. Muizzuddin 2 , A. Neven 1 , R. Sparacio 2 , L. Declercq 1 Glycation is the non-enzymatic reaction between reducing sugars and proteins that leads to the formation of advanced glycation end products (AGEs). In vivo skin autofluorescence ( ex / em = 370/440nm) was used as a non-invasive clinical tool to study skin AGE accumulation in healthy panellists. Using multiple linear regression analysis, it was shown that for panellists below the age of 40, glycation associated in vivo skin fluorescence intensity increased as a function of chronological age and body mass index (BMI). Above the age of 40, the fluorescence was associated to age but not to BMI, suggesting that the effect of age became dominant over BMI. Since the accumulation of AGEs is expected to affect the biomechanical properties of the skin, in vivo skin elasticity data were gathered on a second panel. It was found that skin

	title page
	and D. Maes 2
	1 Estée Lauder Companies, Oevel, Belgium 2 Estée Lauder Companies, Melville, NY, USA * corresponding author Hugo Corstjens Estée Lauder Companies Biological Research Department Europe Nijverheidsstraat 15 2260 Oevel Belgium Tel: +32 14 25 89 04 Fax: +32 14 25 88 60 e-mail: hcorstjens@be.estee.com M A N U S C R I P T
	glycation and elasticity in ageing skin skin ageing, glycation, skin elasticity, body mass index A C C E P T E D
	1

elasticity depended on age and BMI in a similar fashion as to what we observed for the skin fluorescence data. It is hypothesised that skin AGE accumulation contributes to the loss of skin elasticity in aged and/or overweight people.

  more important in the older age group. Based on this analogy it is hypothesised that the accumulation of AGEs as measured with the in vivo skin autofluorescence technique is one of the factors that contributes to the progressive decline in skin elasticity. Similar findings have been presented in a diabetic population in which it was shown that glycation of plantar epidermal proteins could

	seemed to be
	M A N U S C R I P T
	skin elasticity depended on chronological age and BMI in both age groups. For both fluorescence and elasticity, BMI was a statistically significant parameter only in the A C C E P T E D
	group of young panellists (under the age of 40 years). Chronological age affected the
	fluorescence in both age groups but with respect to the elasticity measurements, age
	3

1 Table 1 :

 11 Group characteristics for both clinical studies

Table 2 :

 2 Statistical outcome of the multiple linear regression analysis on the association between in vivo skin fluorescence and skin colour (ITA), chronological age and BMI for the two age groups. Statistical significant parameters are shown in bold. A probability p-value less than 0.05 is deemed statistically significant # Regression coefficient after standardisation of all variables

♦

Table 3 :

 3 Statistical outcome of the multiple linear regression analysis on the association between skin elasticity and chronological age and BMI for the two age groups. Statistical significant parameters are shown in bold.

♦ A probability p-value less than 0.05 is deemed statistically significant # Regression coefficient after standardisation of all variables

A C C E P T E D

M A N U S C R I P T 2 Sasaki, N., Fukatsu, R., Tsuzuki, K., Hayashi, Y., Yoshida, T., Fujii, N., Koike, T., Wakayama, I., Yanagihara, R., Garruto, R., Amano, N., Makita, Z., 1998. Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases. Am. J. Pathol. 153, 1149-1155. Sell, DR., Lane, MA, Johnson, WA., Masoro, EJ., Mock, OB., Reiser, KM., Fogarty, JF., Cutler, RG., Ingram, DK., Roth, GS., Monnier, VM., 1996. Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence. Proc. Natl. Acad. Sci. U.S. A. 93, 485-490. Senolt, L., Braun, M., Olejarova, M., Forejtova, S., Gatterova, J., Pavelka, K., 2005


Bernd Niemann

MD Rolf-Edgar Silber

M.D. Susanne Rohrbach
email: susanne.rohrbach@medizin.uni-halle.de

Age-specific effects of short and long-term caloric restriction on the expression of adiponectin and adiponectin receptors: influence of intensity of food restriction

Keywords: Adiponectin, Adiponectin receptor, caloric restriction, aging, adipose tissue, heart

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M A N U S C R I P T 2

Abstract

Hormonal signals from adipose tissue regulate energy homeostasis and may be involved in anti-aging effects of caloric restriction (CR). The adipokine adiponectin is abundantly expressed in adipose tissue and directly sensitizes the body to insulin. The purpose of the current study was to investigate age-dependent effects of different levels of CR (16%, 2 months or 40%, 6 months) on adiponectin and on its receptors AdipoR1 and AdipoR2 in the left ventricle (LV).

In young and senescent rats, 2 months of moderate CR reduced serum leptin. The same diet was sufficient to enhance serum adiponectin, adiponectin expression (visceral fat) and left ventricular AdipoR1 expression in young but not in senescent rats. The higher degree of CR, however, resulted in a mild induction of adiponectin expression in adipose tissue and release into plasma together with increased LV AdipoR1 also in old rats, while these effects were more pronounced in young rats. These changes in adiponectin activation were associated with reduced LV triglyceride content, suggesting an adiponectin-mediated reduced ectopic lipid deposition in nonadipose tissues. Thus, aging is associated with a loss of adiponectin inducibility by moderate CR. This reduction can only partially be overcome by increasing the degree and duration of CR.

Introduction:

Adiponectin (also termed acrp 30, adipoQ, APM-1, GBP28) is an adipocytokine that is specifically and abundantly expressed in adipose tissue and directly sensitizes the body to insulin. Adiponectin structurally belongs to the complement 1q family (review in [START_REF] Kishore | C1q and tumor necrosis factor superfamily: modularity and versatility[END_REF]) and it exists in oligomeric forms: a low-molecular weight (LMW) trimer, a middle-molecular weight (MMW) hexamer, and high-molecular weight (HMW) 12-to 18mer adiponectin, influencing its biological activity in vivo [START_REF] Pajvani | Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications fpr metabolic regulation and bioactivity[END_REF][START_REF] Pajvani | Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity[END_REF]. The effects of adiponectin seem to be mediated by two distinct receptors: the adiponectin receptors AdipoR1, ubiquitously expressed with highest abundance in skeletal muscle, and AdipoR2, most abundantly expressed in liver [START_REF] Yamauchi | Cloning of adiponectin receptors that mediate antidiabetic metabolic effects[END_REF]. AdipoR1

and AdipoR2 are integral membrane proteins with an internal N-terminus and an external Cterminus [START_REF] Yamauchi | Cloning of adiponectin receptors that mediate antidiabetic metabolic effects[END_REF]. A reduction in the gene expression of these receptors has been described to be associated with diabetes and obesity, while fasting results in renormalization [START_REF] Tsuchida | Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity[END_REF]. In addition, T-cadherin has been suggested to act as an adiponectin-binding protein or coreceptor for an as-yet-unidentified signaling receptor [START_REF] Hug | T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin[END_REF], since it lacks an intracellular domain. AdipoR1 and AdipoR2 serve as receptors for adiponectin and mediate increased activation of the AMP-activated kinase (AMPK), PPARα activities, fatty-acid oxidation, and glucose uptake by adiponectin [START_REF] Yamauchi | Cloning of adiponectin receptors that mediate antidiabetic metabolic effects[END_REF]. Both globular and full-length adiponectin activate AMPK, an enzyme that is switched on by increases in levels of AMP, via increased phosphorylation at Thr-172 of the α-subunit. AMPK functions to restore ATP concentration by stimulating energy-producing processes such as fatty acid oxidation. Activation of AMPK by adiponectin was shown to increase phosphorylation of acetyl-coenzyme A carboxylase (ACC), fatty acid oxidation and glucose uptake via Glut 4, resulting in a decreased triglyceride content and increased insulin sensitivity [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF]. Blockade of AMPK activation by use of a dominant-negative mutant inhibited these effects of full-length or globular adiponectin,

A C C E P T E D M A N U S C R I P T 4
indicating that stimulation of glucose utilization and fatty-acid oxidation by adiponectin occurs through activation of AMPK [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF].

Unlike other adipokines such as TNF-a and resistin, which cause insulin resistance, the expression and circulating levels of adiponectin are reduced in diabetic and obese state in animals as well as in humans. Hypoadiponectinemia has also been demonstrated to be independently associated with the metabolic syndrome, stronger than any other inflammatory marker [START_REF] Matsushita | Comparison of circulating adiponectin and proinflammatory markers regarding their association with metabolic syndrome in Japanese men[END_REF]. Exogenous administration of adiponectin on the other hand improved insulin sensitivity while lowering blood glucose levels in high fat obese mice [START_REF] Yamauchi | The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity[END_REF]. It has been suggested from animal studies that caloric restriction may accomplish the improved insulin sensitivity through modulation of adiponectin levels. Shortterm and long-term caloric restriction (-40%) were reported to result in an increase in adiponectin plasma levels compared to age-matched ad libitum fed rats [START_REF] Zhu | Circulating adiponectin levels increase in rats on caloric restriction: the potential for insulin sensitization[END_REF] as well as in mice [START_REF] Combs | Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin[END_REF]. Similarly, some studies in the human show improved insulin sensitivity together with increased adiponectin plasma concentration following weight reduction by hypocaloric diet [START_REF] Polak | An increase in plasma adiponectin multimeric complexes follows hypocaloric diet-induced weight loss in obese and overweight premenopausal women[END_REF] or by bariatric surgery [START_REF] Guldstrand | Improved beta-cell function after standardized weight reduction in severely obese subjects[END_REF]. Furthermore, adiponectin has been suggested to act as a peripheral "starvation" signal promoting the storage of triglycerides preferentially in adipose tissue thereby reducing ectopic lipid deposition in nonadipose tissue [START_REF] Kim | Obesity-associated improvements in metabolic profile through expansion of adipose tissue[END_REF]. As a consequence, reduced triglyceride levels in liver, skeletal muscle and heart may convey improved systemic insulin sensitivity and improve metabolic syndrome.

Fat cell derived hormones such as adiponectin or leptin can be envisioned as putative mediators of some of the protective effects of caloric restriction. Although long-term caloric restriction is known to retard the aging process in many organisms including mammals, the basic mechanisms of its efficacy remain unclear and its actions in different organs are [START_REF] Masoro | Overview of caloric restriction and ageing[END_REF]. CR results also in a metabolic and transcriptional reprogramming of the adipose tissue and may represent a key mechanism influencing a variety of peripheral organs. While there are many data describing the protective effects of CR on whole body insulin sensitivity mainly related to effects on skeletal muscle physiology, much less is known about changes in cardiac metabolism and cardiac function after application of CR. The duration and intensity of this energy intake restriction without essential nutrient deficiency may have a major impact on this process. Since it is very difficult to differentiate basic primary mechanisms from secondary compensations on the basis of long-term effects of caloric restriction, we applied the lowest degree of restriction (-16% energy intake) and the shortest interval (two months), at which protective actions have been described [START_REF] Merry | Molecular mechanisms linking calorie restriction and longevity[END_REF] in the first group of animals, while the second group of animals was treated for 6 months with a food restriction amounting to -40%. Following this treatment, we investigated the age-dependent effects of different degrees and durations of CR on the expression and release of adiponectin from adipose tissue, the expression of its receptors AdipoR1 and AdipoR2 in left ventricular tissue and the storage of triglycerides in peripheral, adiponectin-dependent organs (LV, liver).

A C C E P T E D M A N U S C R I P T 5 remarkably heterogenous

A C C E P T E D M A N U S C R I P T

6

Methods

Animals and diet protocol

In order to differentiate basic primary mechanisms from secondary compensations on the basis of long-term effects of caloric restriction, we first applied the lowest degree of restriction (-16% energy intake) and the shortest interval (two months), at which protective actions have ever been analyzed in the literature in mammals [START_REF] Merry | Molecular mechanisms linking calorie restriction and longevity[END_REF]. Ad libitum-fed male young and senescent Sprague-Dawley (SD) rats were obtained from Charles River (Germany), caged individually with a light/dark cycle of 12 h and had tap water ad libitum.

Prior to the application of the diet protocols, daily food intake of the normal, ad libitum offered diet of each rat was monitored for 14 days and averaged. In those young rats (4 months at the beginning of the study) on control diet (Altromin R 1344/1800; 1800 cal/g, CD)

the daily energy intake was 52.5±3.8 kcal, in young rats on -16% caloric restriction (Altromin R 1344/1500; 1500 cal/g, CR) this daily intake was 45.2±0.7 kcal. In old rats (22-24 months at the beginning of the study) the daily energy intake amounted to 46.4±5.0 kcal in control diet and to 39.9±0.2 kcal in -16% caloric restricted rats.

In a second group of animals, rats were randomly assigned to either receive control diet or a calorically restricted diet for 6 months. During the diet protocol, rats on control diet (Altromin R 1344/2500; 2505 cal/g, CD) received their individual prediet average, but not more, in order to avoid any degree of diet-induced obesity. Rats subjected to caloric restriction received also their prediet average, but of a caloric reduced, fibre-rich diet (Altromin R 1344/1500; 1500 cal/g; CR). Thus, in young rats (4 months at the beginning of the study) on control diet for 6 months the daily energy intake was 64.8±2.2 kcal and in young rats on -40% caloric restriction this daily intake was 37.8±1.9 kcal. In old rats (22-24 months at the beginning of the study) the daily energy intake amounted to 57.9±1.8 kcal in control diet and to 36.6±0.1 kcal in -40% caloric restricted rats. All animals (group 1 and group 2)

A C C E P T E D M A N U S C R I P T 7
were fasted for 12 hours before killing and blood was obtained by aortic puncture. All treatments of animals throughout this study received local institutional animal care and use committee approval.

RNA Extraction

Total RNA was isolated from frozen visceral fat and from left ventricular (LV) tissue by guanidine thiocyanate/cesium chloride centrifugation. Integrity and quality of the RNA was confirmed by agarose gel electrophoresis and the concentration determined by measuring UVabsorption.

RT-PCR

Reverse transcription (RT) of RNA samples was carried out for 30 minutes at 42°C. Realtime PCR and data analysis were performed using the Mx3000P Multiplex Quantitative PCR System (Stratagene). DNA amplification was performed as follows: initial denaturation at 95°C for 10 minutes, 40 cycles of amplification (denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 60 seconds), followed by a denaturation at 95°C for 60 seconds and a melting curve over the range from 55°C up to 95°C. Fluorescence data were collected at the end of the annealing stage of amplification. We performed Real-time PCR of adiponectin (forward 5'-AATCCTGCCCAGTCATGAAG-3; reverse 5'-CATCTCCTGGGTCACCCTTA-3'), AdipoR1 (forward 5'-CTTCTACTGCTCCC CACAGC-3'; reverse 5'-GACAAAGCCCTCAGCGATAG-3'), AdipoR2 (forward 5'-TAC-ACACAGAGACGGGCAAC-3'; reverse 5'-GCAGTACACCGTGTGGAAGA-3') and 18S

rRNA (18S rRNA Control kit, Yakima Yellow®-Eclipse® Dark Quencher, Eurogentec) in samples derived from rat adipose tissue or left ventricles. Each assay was performed in

A C C E P T E D M A N U S C R I P T 8
duplicate and validation of PCR-runs was assessed by evaluation of the melting curve. All data of mRNA are given as relative units of 18S rRNA concentrations.

Western blotting

Frozen LV tissue was rapidly homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodiumdeoxycholate and protease inhibitor cocktail (Sigma). Proteins were quantified using the BCA Protein Assay (Pierce). 50µg of protein were loaded on a 10% SDS-PAGE gel. After electrophoresis, proteins were transfered to a nitrocellulose membrane. The filters were blocked and then incubated with antibodies directed against adiponectin (BioVendor, diluted 1:1000), AdipoR1, AdipoR2 (both Abcam, 1:1000, Vimentin (Sigma, 1:2000) as well as GAPDH (Abcam, 1:5000). After incubation with peroxidase-conjugated secondary antibody, blots were subjected to the enhanced chemiluminescent detection method (Amersham) and exposed to a Kodak MR film.

Analyses of Plasma Adiponectin, Plasma Leptin and Tissue Triglycerides

Serum adiponectin and leptin concentrations were measured by using commercial Enzyme-Linked-Immunosorbent-Assays (Mouse/Rat Adiponectin ELISA-kit, B-Bridge International Inc.; Mouse/Rat Leptin ELISA-Kit, BioVendor) according to the manufacturers' instructions.

Tissue triglycerides were extracted as described before (Bligh and Dyer 1959) and measurement was performed by an enzymatic colorimetric method using a commercial kit (GPO Trinder, Sigma).

Statistical Analysis

All values are expressed as mean ± SEM. Statistical analysis of differences observed between the groups was obtained by ANOVA. Statistical significance was accepted at the level of p<0.05.

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9

Results

Systemic effects of 16% and 40% caloric restriction

In the moderate, short-term restriction group, the body weight of young animals under control diet increased by 17 ± 1% within two months, while 16% caloric restriction attenuated this increase to 9 ± 1% (Fig. 1A). In the old control rats, however, body weight decreased by 9 ± 1 % and this decline was augmented to an 18 ± 1% weight loss in the animals under 16% caloric restriction for 2 months (Fig. 1A). There was no difference in the daily water intake among the rats. Ventricular weights were higher in old control rats than in young ones, also after normalization per body weight (Tab. 1). However, the moderate caloric restriction of two months duration did not alter ventricular weight/body weight ratios within the agematched groups (Tab. 1)

In the young, growing animals under control diet, body weight increased by 10 ± 1% within six months, while 40% caloric restriction resulted in a decrease by 14 ± 2% (Fig. 1C). In the old control rats, body weight was decreased by 10 ± 2% after 6 months, and this decline was augmented to 38 ± 4% weight loss in the animals under caloric restriction (Fig. 1C). None of the young animals died during the study (not shown), while survival was significantly increased in old animals under 40% caloric restriction within the first 4 months of the diet compared to old animals under control diet (Fig. 1D). Ventricular weights were higher in old control rats than in young ones (Tab. 2) and were significantly decreased under 40% caloric restriction in young as well as in old animals (Tab. 2).

Effects of caloric restriction on the expression and release of adiponectin

The moderate caloric restriction significantly enhanced the circulating levels of the adipocytederived hormone adiponectin in young rats (Fig. 2A), but it did not modify adiponectin plasma levels in the old rats (Fig. 2A), illustrating a hitherto unidentified loss of reactivity in adipose tissue with advanced age. On the other hand, the restriction induced the anticipated

A C C E P T E D M A N U S C R I P T
10 decline in plasma leptin only in the old rats, while this decline did not reach significance in the young rats (Fig. 2B). 40% caloric restriction resulted in a dramatic increase in adiponectin plasma levels in young animals (Fig. 2C) as has been observed previously with caloric restriction or fasting in animals [START_REF] Zhu | Circulating adiponectin levels increase in rats on caloric restriction: the potential for insulin sensitization[END_REF]. Old control animals on the other hand demonstrated an adiponectin deficit and even the strong stimulation with 40% caloric restriction resulted only in a mild increase of adiponectin plasma concentrations in these animals (Fig. 2C). These differences in adiponectin plasma levels were also reflected by adiponectin mRNA and protein expression in the visceral adipose tissue: 16% and 40% of caloric restriction resulted in a strong induction of adiponectin expression in young animals (Fig. 3). Old control animals on the other hand demonstrated an adiponectin deficit. While 16% of caloric restriction were not sufficient to stimulate the visceral adipose tissue of senescent animals (Fig. 3A), the strong stimulation with 40% caloric restriction resulted in a modest increase of adiponectin mRNA and protein expression in these animals (Fig. 3B).

Leptin plasma levels were reduced in both groups under 40% caloric restriction (Fig. 2D).

Effects of caloric restriction on the expression of adiponectin receptors

Adiponectin influences cellular gene expression and signal transduction by binding to the receptors AdipoR1 and AdipoR2. The expression of AdipoR1 mRNA was higher than the expression of AdipoR2 mRNA in left ventricular tissue (Fig. 4). While AdipoR2 expression was not altered following moderate or strong caloric restriction in any of the groups, the higher degree of caloric restriction (-40%) resulted in a strong induction of AdipoR1 mRNA and protein in young and in old animals (Fig. 4A), that was not observed in the old hearts following 16% of CR (Fig. 4B). The corresponding values from AdipoR1 Western blot analyses of the LV after 16% CR are: in young rats 147.6±14.9 vs. 292. A C C E P T E D M A N U S C R I P T 11 protein expression was not different among any of the groups (not shown). We observed no significant age-dependent decrease in the expression of LV adiponectin receptors as described in the skeletal muscle of mice suffering from diabetes or obesity [START_REF] Tsuchida | Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity[END_REF].

Effects of caloric restriction on the tissue triglyceride content

Recently, adiponectin has been suggested to act as a peripheral "starvation" signal promoting the storage of triglycerides preferentially in adipose tissue thereby reducing triglyceride levels in the liver and muscle [START_REF] Kim | Obesity-associated improvements in metabolic profile through expansion of adipose tissue[END_REF]. Left ventricular content of triglycerides was increased in senescent animals and did not decrease after 2 months of moderate caloric restriction (4.87 mg/g wt ± 1.19 vs. 4.74 mg/g wt ± 1.23) while it was significantly lowered by the same caloric restriction in young animals (2.90 mg/g wt ± 1.29 vs. 1.64 mg/g wt ± 0.63, p<0.01). Similar changes were observed in the liver of these animals (5.51 mg/g wt ± 0.89 vs. 4.99 mg/g wt ± 0.53 in senescent animals; 4.46 mg/g wt ± 0.43 vs. 3.07 mg/g wt ± 0.30 in young animals, p<0.05). The higher degree and longer lasting caloric restriction (-40%, 6 months) resulted in a significant decrease in left ventricular triglyceride content in young (2.61 mg/g wt ± 0.36 vs. 1.70 mg/g wt ± 0.28, p<0.05) and in senescent animals (4.69 mg/g wt ± 0.70 vs. 2.76 mg/g wt ± 0.32). Similar changes were observed in the liver of these animals (not shown).

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12

Discussion

Caloric restriction is a reproducible and simple experimental manipulation known to extend maximum lifespan and it has also been shown to retard a variety of age-associated pathophysiological changes [START_REF] Masoro | Overview of caloric restriction and ageing[END_REF]. Aging is associated with insulin resistance and alterations in body fat distribution [START_REF] Enzi | Subcutaneous and visceral fat distribution according to sex, age, and overweight, evaluated by computed tomography[END_REF]. Among others, CR retards these ageassociated changes in insulin sensitivity, possible through decreasing obesity [START_REF] Barzilai | The role of fat depletion in the biological benefits of caloric restriction[END_REF]. Accordingly, surgical removal of visceral fat has been shown to result in a comparable improvement of insulin resistance [START_REF] Barzilai | Surgical removal of visceral fat reverses hepatic insulin resistance[END_REF]. The fat cell derived hormone adiponectin has been suggested to play a key role in this process by different mechanisms such as increased glucose uptake, altered fatty acid combustion [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] or decreased ectopic lipid deposition [START_REF] Kim | Obesity-associated improvements in metabolic profile through expansion of adipose tissue[END_REF]. Similarly, some studies in the human show improved insulin sensitivity together with increased adiponectin plasma concentration following weight reduction by hypocaloric diet [START_REF] Polak | An increase in plasma adiponectin multimeric complexes follows hypocaloric diet-induced weight loss in obese and overweight premenopausal women[END_REF] or by bariatric surgery [START_REF] Guldstrand | Improved beta-cell function after standardized weight reduction in severely obese subjects[END_REF].

However, the longevity enhancing action of caloric restriction appears to depend on the degree of restriction, up to a reduction down to 35% of ad libitum caloric intake, and a duration of two months is regarded as minimum required for metabolic equilibrium [START_REF] Merry | Molecular mechanisms linking calorie restriction and longevity[END_REF]. A reduction down to 84%, as applied in the short-term low degree study here, is below the threshold range of restriction with documented survival benefit in rodents [START_REF] Merry | Molecular mechanisms linking calorie restriction and longevity[END_REF].

A comparative analysis on effects of fasting, short-term or long-term CR on gene expression of white adipose tissue in young mice showed that only a few genes were altered by fasting or short-term CR, making these transcript phenotypes almost undistinguishable from control animals [START_REF] Higami | Adipose tissue energy metabolism: altered gene expression profile of mice subjected to longterm caloric restriction[END_REF]. Long-term CR (9 months) on the other hand resulted in major changes in gene expression including genes involved in amino acid, lipid, carbohydrate or mitochondrial energy metabolism as well as genes associated with altered insulin sensitivity

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13 [START_REF] Higami | Adipose tissue energy metabolism: altered gene expression profile of mice subjected to longterm caloric restriction[END_REF]. Long-term CR also reduced the size and cytoplasmic lipid content of adipocytes and up-regulated genes involved in adipocyte differentiation [START_REF] Higami | Adipose tissue energy metabolism: altered gene expression profile of mice subjected to longterm caloric restriction[END_REF].

Unfortunately, age-associated transcriptionally differences in the response to fasting, shortterm or long-term CR were not investigated in that study [START_REF] Higami | Adipose tissue energy metabolism: altered gene expression profile of mice subjected to longterm caloric restriction[END_REF]. The present study shows that the intensity and duration of caloric restriction determines its age-dependent effects on adipose tissue and cardiac muscle. While moderate CR was sufficient to induce potentially protective effects only in young animals, the higher intensity and duration of CR evoked similar, although somewhat weaker effects also in the old animals. Therefore, therapeutics that influence insulin sensitivity or mimic some of the effects of CR may have tremendously different impact on patients depending on their age.

Two months of -16% caloric restriction attenuated the adolescence-associated gain in body weight in young animals (Fig. 1A) while the higher degree of CR (6 months, -40%) resulted in a weight loss also in young animals (Fig. 1C). This was also associated with an improved survival in senescent rats (Fig. 1D) after 4-6 months, which was not achieved by the moderate, short-term CR (Fig. 1B). Adiponectin is a key hormone to control body metabolism as it was shown to increase fatty acid oxidation and glucose uptake, to decrease triglyceride content and to increase insulin sensitivity [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF]. Therefore, adiponectin expression in adipose tissue and release into plasma may have a major impact on the protective and insulin sensitizing effects of caloric restriction. Furthermore, weight loss has been shown to result in an increase of adiponectin expression in visceral adipose tissue in lean and obese rats [START_REF] Milan | Resistin and adiponectin expression in visceral fat of obese rats: effect of weight loss[END_REF] and was reported to result in increased adiponectin plasma levels in rats after caloric restriction compared to age-matched ad libitum fed animals [START_REF] Zhu | Circulating adiponectin levels increase in rats on caloric restriction: the potential for insulin sensitization[END_REF]. Both types of CR significantly enhanced the circulating levels of the adipocytederived hormone adiponectin in young rats (Fig. 2 A+C). However, the moderate restriction did not modify adiponectin plasma levels in old rats (Fig. 2A). The higher intensity and longer

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14 duration of CR further potentiated the release of adiponectin from adipose tissue in young animals, while it resulted only in a modest induction in old animals, which is still lower than in young controls (Fig. 2C). This illustrates a loss of responsivity in adipose tissue with advanced age, although Combs et al. reported that chronic caloric restriction (-40%) results in an increase in adiponectin plasma levels in young as well as in senescent mice [START_REF] Combs | Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin[END_REF]. However, this loss of responsivity did not include alterations in leptin plasma changes following CR (Fig 2B+D). As described previously in Fischer 344 rats by others [START_REF] Mooradian | Age-related changes in plasma leptin binding activity in rats: A comparison of a simple acid-ethanol precipitation technique with column chromatography[END_REF] serum leptin concentration was increased in senescent rats. Both types of caloric restriction resulted in a decrease in serum leptin that was more pronounced following the high degree CR in young but not in old animals (Fig. 2B+D). Similar to adiponectin plasma levels, adiponectin mRNA and protein expression were induced in visceral fat of young rats (Fig. 3) following both types of diet. Old animals, however, critically depend on a stronger stimulation (40% CR for 6 months) to upregulate their adiponectin mRNA and protein expression in visceral adipose tissue (Fig. 3B).

The expression of both adiponectin receptors has been shown to be increased in fasted animals [START_REF] Tsuchida | Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity[END_REF], in humans after 4 weeks of physical activity [START_REF] Bluher | Circulating adiponectin and expression of adiponectin receptors in human skeletal muscle: associations with metabolic parameters and insulin resistance and regulation by physical training[END_REF] but decreased in refed mice [START_REF] Tsuchida | Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity[END_REF]. Whereas the cardiac muscle of young animals demonstrated a diet-induced induction of AdipoR1 receptor (Fig. 4) as described before in the skeletal muscle of mice [START_REF] Tsuchida | Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity[END_REF], no increase is observed in senescent animals following the moderate restriction (Fig. 4). 6 months of stronger CR resulted in a comparable induction of AdipoR1 in the cardiac muscle of young and old animals. Cardiac AdipoR2 was not altered in any group (Fig. 4). This indicates a remarkable difference to the effects of CR in the LV compared to skeletal muscle, since we observed an induction of AdipoR1 and AdipoR2 in young animals in this organ after CR before [START_REF] Rohrbach | Age-associated loss in adiponectin-activation by caloric restriction: Lack of compensation by enhanced inducibility of adiponectin paralogs CTRP2 and CTRP7[END_REF]. Although AdipoR1 or AdipoR2 are mainly expressed in

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15 cardiomyocytes [START_REF] Fujioka | Role of adiponectin receptors in endothelin-induced cellular hypertrophy in cultured cardiomyocytes and their expression in infarcted heart[END_REF] other cell types among them macrophages, endothelial cells or smooth muscle cells [START_REF] Chinetti | Expression of adiponectin receptors in human macrophages and regulation by agonists of the nuclear receptors PPARalpha, PPARgamma, and LXR[END_REF] have also been shown to express the adiponectin receptors. Therefore, we can not exclude that these cellular sources may have contributed to the increased AdipoR1 mRNA and protein expression in our LV lysates. One can speculate that the adiponectin deficit in old animals may be further amplified in peripheral organs including the heart by the absence of inducibility of the adiponectin receptors AdipoR1

and AdipoR2 in the senescent heart following moderate caloric restriction (Fig. 4). Thus, potential cardioprotective effects of the adiponectin axis ranging from improved myocardial glucose and lipid metabolism or protection against ischemia/reperfusion injury [START_REF] Tao | Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress[END_REF] to improved vascular function by modulating the endothelial inflammatory response [START_REF] Goldstein | Adiponectin: A novel adipokine linking adipocytes and vascular function[END_REF]) may be blunted in senescent hearts. The expression of both AdipoR1 and AdipoR2 was also shown to be significantly reduced in skeletal muscle and adipose tissue of ob/ob mice together with hyperinsulinemia and impairment of adiponectinmediated activation of AMPK [START_REF] Tsuchida | Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity[END_REF] -a state that one could call adiponectin resistance. However, senescent animals have not been investigated so far and there are also reports in the literature arguing against a direct responsiveness of AdipoR1/2 to changes in the nutritional status [START_REF] Beylot | Expression of the adiponectin receptors AdipoR1 and AdipoR2 in lean rats and in obese Zucker rats[END_REF]. Although there are no data available in the literature describing changes in the expression of adiponectin receptors in cardiac or skeletal muscle, it has been described that very short-term (3 days) CR differentially triggers mitochondrial adaptive mechanisms in the skeletal muscle depending on the intensity of food restriction [START_REF] Johnson | Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats[END_REF]. Thus, adipose tissue [START_REF] Higami | Adipose tissue energy metabolism: altered gene expression profile of mice subjected to longterm caloric restriction[END_REF]) and muscular tissue [START_REF] Johnson | Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats[END_REF] both show a graduate response to the stimulatory effects of different types of CR. Unfortunately, both studies [START_REF] Higami | Adipose tissue energy metabolism: altered gene expression profile of mice subjected to longterm caloric restriction[END_REF][START_REF] Johnson | Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats[END_REF] did not investigate senescence-associated alterations in this response.

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Adiponectin acts as a "starvation" signal in peripheral organs such as liver, skeletal muscle or cardiac muscle by promoting the storage of triglycerides preferentially in adipose tissue while the ectopic lipid deposition in nonadipose tissues is reduced [START_REF] Kim | Obesity-associated improvements in metabolic profile through expansion of adipose tissue[END_REF]. Similar to the results obtained for the adiponectin expression and release into the plasma, senescent rats did not show a significant decrease of cardiac (and liver) triglycerides in response to the stimulatory effects of moderate caloric restriction as described before in kidneys from senescent Fischer 344 rats [START_REF] Jiang | Calorie restriction modulates renal expression of sterol regulatory element binding proteins, lipid accumulation, and age-related renal disease[END_REF], while the stronger stimulus (6 months, -40%)

was sufficient in old and in young animals. Others have also shown that 1 week of moderate caloric restriction in young rats did not affect the triglyceride content while after 3 weeks muscular but not liver triglyceride content was reduced (Barazzoni et al., 2005). No data were obtained from senescent animals in that study (Barazzoni et al., 2005). However, besides adiponectin other adipokines or hormones can be envisioned as regulators of lipid and energy metabolism reacting to caloric restriction. Ghrelin, which was not investigated in the current study, for example has been shown to induce tissue-specific changes in lipid metabolism favouring triglyceride deposition in liver over skeletal muscle (Barazzoni et al., 2005).

Alterations in the type of energy substrate or the rates of ATP production have consequences on LV contractile function and stress responsivity of the heart. Novel mechanisms for heart disease in obesity, called "lipotoxic cardiomyopathy", whereby lipid accumulation within the myocardium is directly cardiotoxic and causes LV dysfunction were described in rodents and subsequently also reported in patients with congestive failure [START_REF] Szczepaniak | Forgotten but not gone: the rediscovery of fatty heart, the most common unrecognized disease in America[END_REF][START_REF] Unger | Lipotoxic diseases[END_REF]. Recent studies in healthy individuals and patients with heart failure reveal that myocardial lipid content increases with the degree of obesity and contributes to the progressive deterioration in LV function observed in obese patients [START_REF] Szczepaniak | Forgotten but not gone: the rediscovery of fatty heart, the most common unrecognized disease in America[END_REF].

Changes in the expression and release of adipose-derived cytokines such as adiponectin may contribute to this ectopic storage of triglycerides [START_REF] Kim | Obesity-associated improvements in metabolic profile through expansion of adipose tissue[END_REF] in the LV with subsequent functional deterioration.

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17 So far, there are only few studies investigating the age-specific effects of caloric restriction on cardiac function and cardiac metabolism including triglyceride storage within the cardiomyocyte. Ischemic preconditioning has no beneficial effect on the postischemic functional recovery of senescent human myocardium [START_REF] Bartling | Cardio-protective determinants are conserved in aged human myocardium after ischemic preconditioning[END_REF], while it has also been shown to reduce postischemic dysfunction in the hearts from young-adult and foodrestricted but not in ad libitum-fed senescent rats [START_REF] Abete | Cardioprotective effect of ischemic preconditioning is preserved in food-restricted senescent rats[END_REF]. Therefore, mechanisms activated by caloric restriction itself or by CR mimetics may represent powerful tools to improve cardiac metabolism and to preserve cardiac functions also in aged human myocardium. The fat cell derived hormone adiponectin has been shown to mediate various cardioprotective effects and can therefore be envisioned as one putative mediator of the protective effects of caloric restriction on the heart. Our study provides data that senescent animals display a significant adiponectin deficit and only high-degree caloric restriction may improve the release of adiponectin from adipose tissue although on a very low level. This observation may also have an impact on therapeutical strategies or outcome of elderly patients suffering from diseases such as type 2 diabetes, obesity, metabolic syndrome or cardiac dysfunction. CR is feasible in laboratory animals and has proven to be a valuable research tool, but it is not applicable in the aging population as a therapeutic or preventive intervention because of the enhanced risks for sarcopenia, osteoporosis and depression. Nevertheless, cardiometabolic pathways activated during CR might yield targets for pharmacological mimetics of selected components within the spectrum of CR actions.

Figure 1 :

 1 Figure LegendsFigure 1: Effects of caloric restriction on body weight and survival 1A: The gain in body weight is reduced in young rats under 16% caloric restriction (n = 12;

Figure 2 :

 2 Figure 2: Effects of caloric restriction on plasma concentrations of adiponectin and leptin 2A+B: Two months of 16% caloric restriction raise adiponectin serum levels in young rats

Figure 3 :

 3 Figure 3: Effects of caloric restriction on adiponectin mRNA and protein expression in visceral fat A: Data from 2 months 16% CR; B: Data from 6 months, 40% CR.

Figure 4 :

 4 Figure 4: Effects of caloric restriction on AdipoR1 and AdipoR2 mRNA and protein expression in the LV A: Data from 2 months 16% CR; B: Data from 6 months, 40% CR.

  Figure1

Table 1 : Effects of 16% caloric restriction on LV and body weight

 1

	Left ventricle (g)	diet CD	young rats 0.82 ± 0.03	n old rats 7 1.37 ± 0.06 a	n
		CR	0.74 ± 0.01	12	1.29 ± 0.03 a
	Body weight before diet (g)	CD	332 ± 6	7	556 ± 6 a
		CR	334 ± 4	12	568 ± 6 a
	Body weight after diet (g)	CD	396 ± 7	7	509 ± 11 a
		CR	362 ± 4 *	12	466 ± 5 ***, b
	LV/BW ( 0 / 00 )	CD CR	2.13 ± 0.06 2.03 ± 0.02	7 12	2.72 ± 0.17 a 2.76 ± 0.07 a

Acknowledgements

We appreciate the technical assistance of R. Busath, R. Gall and B. Heinze. This study was supported by the DFG (RO 2328/2-1), Deutsche Stiftung für Herzforschung (F /05/ 05),