Paola Venditti
email: venditti@unina.it

A Bari

L Di Stefano

A Cardone

F Della

S Di Meo

Involvement of PGC-1, NRF-1, and NRF-2 in metabolic response by rat liver to hormonal and environmental signals

Keywords: Cold-exposure, Thyroid hormone, Mitochondrial respiration, Mitochondrial proliferation, Transcription factor activators

We studied liver oxidative capacity and O 2 consumption in hypothyroid rats treated for 10 days with T 4 , or T 3 , or treated for 10 days with T 3 and exposed to cold for the last two days. The metabolic response of homogenates and mitochondria indicated that all treatments increased the synthesis of respiratory chain components, whereas only the cold induced mitochondrial proliferation. Determination of mRNA and protein expression of transcription factor activators, such as NRF-1 and NRF-2, and coactivators, such as PGC-1, showed that mRNA levels, except PGC-1 ones, were not related to aerobic capacities. Conversely, a strong correlation was found was between cytochrome oxidase activity and PGC-1 or NRF-2 protein levels. Such a correlation was not found for NRF-1. Our results strongly support the view that in rat liver PGC-1 and NRFs are responsible for the iodothyronine-induced increases in respiratory chain components, whereas their role in cold-induced mitochondrial proliferation needs to be further on clarified.

Observations made in clinical and experimentally induced hyperthyroidism have shown that the main biological effect of thyroid hormone is to accelerate energy expenditure. In fact, the development of a hyperthyroid state in vertebrates leads to an increase in their metabolic rate due to an enhanced rate of O 2 consumption in most of body tissues [START_REF] Barker | Metabolism of tissues excised from thyroxine-injected rats[END_REF]. Although thyroxine (T 4 ) is the main product of thyroid secretion is widely shared the view that T 4 is only a prohormone and must be activated by deiodination to triiodothyronine (T 3 ) in order to initiate thyroid hormone action. Thus, long-term effects of thyroid hormones or states modulating thyroid gland activity, such as cold exposure, are believed to be transduced through T 3 -induced changes in both nuclear and mitochondrial gene expression. In liver from both T 3 -treated [START_REF] Fernández | Superoxide radical generation, NADPH oxidase activity, and cytochrome P-450 content of rat liver microsomal fraction in an experimental thyroid state: relation to lipid peroxidation[END_REF][START_REF] Seitz | Rapid thyroid hormone effect on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell[END_REF]Venditti et al., 2006a) and cold-exposed animals [START_REF] Guernsey | The cell membrane sodium pump as a mechanism for increasing thermogenesis during cold acclimation in rats[END_REF][START_REF] Shiota | Effect of norepinephrine on gluconeogenesis in perfused livers of cold-exposed rats[END_REF][START_REF] Venditti | Cold-induced hyperthyroidism produces oxidative damage in rat tissues and increases susceptibility to oxidants[END_REF] such changes lead to enhanced tissue O 2 consumption. However, in experimental hyperthyroidism the increase in liver respiration involves increases in the amount of respiratory chain proteins and inner surface area [START_REF] Jacovcic | Biochemical and stereological analysis of rat liver mitochondria in different thyroid states[END_REF] of mitochondria without changes in their number [START_REF] Goglia | The effect of thyroid state on respiratories activities of three rat liver mitochondrial fractions[END_REF] and protein mass (Venditti, et al., 2006a).

Conversely, in cold-induced functional hyperthyroidism the increase in liver respiration involves proliferation of mitochondria [START_REF] Goglia | Thyroid state and mitochondrial population during cold exposure[END_REF] associated with an enhancement in tissue content of mitochondrial proteins [START_REF] Venditti | Cold-induced hyperthyroidism produces oxidative damage in rat tissues and increases susceptibility to oxidants[END_REF]Venditti et al., 2006a).

The above results suggest that T 3 is responsible for the changes in hepatic content of respiratory chain components elicited by cold exposure and hormonal treatment. Conversely, other factors, exhibiting different serum concentrations in functional and experimental hyperthyroidism, should be involved in mitochondrial proliferation associated to cold exposure. Unlike cold exposure, T 3 administration strongly decreases serum levels of T 4 , (Venditti et al., 2006a), which has been reported to have intrinsic biological activity in the cold [START_REF] Cageao | Effects of thyroid hormones on mitochondrial oxygen consumption in brown adipose tissue and heart from cold exposed hypothyroid rats[END_REF]. Plasma levels of catecholamines, which are not modified in altered thyroid states [START_REF] Stoffer | Plasma catecholamines in hypothyroidism and hyperthyroidism[END_REF], remarkably increase during cold exposure [START_REF] Storm | Thyroid hormone-catecholamines interrelationship during exposure to cold[END_REF]. Furthermore, a fall in liver mitochondria respiration is elicited by injection of adrenergic-receptor blockers to cold acclimated rats [START_REF] Zaninovich | Mitochondrial respiration in muscle and liver from cold-acclimated hypothyroid rats[END_REF].

In a previous work we used euthyroid rats to examine liver metabolic response to treatments, which differentially affect circulating T 4 levels (Venditti et al., 2006a). Despite some supporting results, the idea A c c e p t e d M a n u s c r i p t that thyroxine can play a role in liver response to cold remains still to demonstrate. Therefore, in the present work we compared the effects on liver oxidative metabolism of 10 days of T 3 or T 4 treatment to rats made hyperthyroid by PTU and iopanoic acid to block the deiodinase activities [START_REF] Obregon | Evidence against a major role of L-thyroxine at the pituitary level: studies in rats treated with iopanoic acid (telepaque)[END_REF]. To shown possible involvement of catecholamines in cold-induced metabolic changes, we also studied the effects of 2 day cold exposure (4° C) on T 3 -treated hypothyroid rats. Moreover, metabolic responses were related to changes in the expression levels of nuclear respiratory factors-1 and -2 (NRF-1 and NRF-2) and nuclear receptor co-activator PGC-1, which are regulated by thyroid hormone [START_REF] Weitzel | Two thyroid hormone-mediated gene expression patterns in vivo identified by cDNA expression arrays in rat[END_REF] and play a role in the mitochondrial biogenesis [START_REF] Scarpulla | Nuclear activators and coactivators in mammalian mitochondrial biogenesis[END_REF].

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

Materials and methods

Materials

All chemicals used (Sigma Chimica, Milano, Italy) were of the highest grades available. Response to oxidative stress was determined by using reagents and instrumentation of the commercially available Amerlite System (Ortho-Clinical Diagnostics, Milano, Italy). Serum levels of free triiodothyronine (FT 3 ) and thyroxine (FT 4 ) were determined by using commercial RIA kits (DiaSorin, Salluggia, Italy).

Animals

The experiments were carried out on 70-day-old male Wistar rats, supplied by Nossan (Correzzana, Italy) at day 45 of age. From day 49, animals were randomly assigned to one of four groups: hypothyroid rats (H), hypothyroid rats made hyperthyroid by T 3 (HT 3 ) or T 4 (HT 4 ) treatment, hypothyroid rats made hyperthyroid by T 3 treatment and exposed for 2 days to cold (HT 3 +CE). In H rats, both thyroid and deiodinase activities were chronically inhibited by i.p. administration of PTU (1 mg/100 g body weight, once per day for 3 weeks), together with administration of iopanoic acid (IOP) given to 10, 13, 16, 19, and 21 days after the first PTU injection. The other rats, which undergone the same treatment of H rats were also intraperitoneally administered with T 4 (HT 4 ) or with T 3 (HT 3 and HT 3 +CE) (10 μg/100g body weight, once per day for 10 days before sacrifice), and exposed to 41°C for 2 days before sacrifice (HT 3 +CE). H, HT 3 , and HT 4 rats were kept at room temperature of 24 1°C. The combined treatment with PTU and IOP inhibits both the production of thyroid hormones and all deiodinase enzymes. Thus, it excludes the possibility that the effects observed following the administration of iodothyronines can be attributed to any of their deiodinated products and cold exposure can change thyroid hormone levels. The rats were exposed to cold for two days because a such period is sufficient to induce remarkable mitochondrial proliferation [START_REF] Venditti | Cold-induced hyperthyroidism produces oxidative damage in rat tissues and increases susceptibility to oxidants[END_REF].

All rats were kept under the same environmental conditions and were provided with water ad libitum and commercial rat chow diet (Nossan).

The treatment of animals in these experiments was in accordance with the guidelines set forth by the University's Animal Care Review Committee.

Tissue preparation

The animals were sacrificed by decapitation while under ether anaesthesia. Arterial blood samples were collected and later analyzed to determine plasma levels of FT 3 and FT 4 by radioimmunoassay. Liver was rapidly excised and placed into ice-cold homogenization medium (HM) (220 mM mannitol, 70 mM sucrose, 1 mM EDTA, 0.1% fatty acid-free albumin, 10 mM Tris, pH 7.4). Then, the tissue was weighed, finely minced, and washed with HM. Finally, liver fragments were gently homogenized (20% w/v) in HM using a glass Potter-Elvehjem homogenizer set at a standard velocity (500 rpm) for 1 min. Aliquots of liver homogenates were used for analytical procedures and preparation of mitochondrial fractions.

Preparation of mitochondrial fraction

The homogenates, diluted 1:1 with HM, were freed of debris and nuclei by centrifugation at 500 g for 10 min at 4°C. The resulting supernatants were centrifuged at 10,000 g for 10 min. The mitochondrial pellets were washed twice with isolation medium (IM) (220 mM mannitol, 70 mM sucrose, 1 mM EGTA, 20 mM Tris, pH 7.4), resuspended in the same solution and used for determination of analytical procedures.

The protein content of liver preparations was determined, upon solubilization in 0.5% deoxycholate, by the biuret method [START_REF] Gornall | Determination of serum proteins by mean of the biuret reaction[END_REF] with bovine serum albumin as standard.

Oxygen consumption

Oxygen consumption of liver homogenates was monitored at 30° C by a Gilson respirometer in 1.6 ml of incubation medium (145 mM KCl, 30 mM Hepes, 5 mM KH 2 PO4, 3 mM MgCl 2 , 0.1 mM EGTA, pH 7.4) with 50 μl of homogenate and succinate (10 mM), plus 5 μM rotenone (Rot), or pyruvate/malate (10/2.5 mM) as substrates, in the absence (State 4) and in the presence (State 3) of 500 μM ADP. Mitochondrial State 4 and State 3 respiration was monitored by the same method using 0.25 mg of mitochondrial protein per ml in the presence of Complex I-and Complex II-linked substrates. The first three complexes of the electron transport system were measured by spectrophotometric methods [START_REF] Ragan | Sub-fractionation of mitochondria and isolation of the proteins of oxidative phosphorylation[END_REF] using a Beckman (Fullerton CA USA) model DU 640. Complex IV (cytochrome c oxidase) activity was determined by a polarographical procedure at 30° C [START_REF] Barré | Increased oxidative capacity in skeletal muscles from acclimated ducklings: A comparison with rats[END_REF] using a Gilson glass respirometer equipped with a Clark oxygen electrode (Yellow Springs Instruments Ohio USA).

Activity of complexes of respiratory chain

Cytochrome oxidase activity and mitochondrial protein content

Cytochrome c oxidase (COX) activity of homogenates was also determined and the ratio between the cytochrome oxidase activities in homogenates and mitochondria supplied rough estimates of hepatic content of mitochondrial proteins. Moreover, because the in vitro activity of COX is positively correlated to the maximal oxygen consumption [START_REF] Simon | Relationship of cytochrome oxidase activity to vertebrate total and organ oxygen consumption[END_REF], it was also used as a measure of the aerobic metabolic capacity of tissues.

Mitochondrial protein SDS-PAGE

SDS-PAGE was performed according to Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF], using a 10% acrylamide as resolving gel. Samples were prepared by diluting 10 μl of mitochondrial suspension containing 1.5 mg/ml of protein with 5 μl of 3% SDS, 30% glycerol, 15% b-mercaptoethanol 0.1 % bromophenol blue, 0.187 M Tris base, pH 6.8 and were boiled for 5 min before loading on the gel. Gel was run in the mini protean equipment (Bio-Rad) for about 1 hr at constant voltage (25 V). The gel upon electrophoresis was fixed with 25 % isopropanol-10 % acetic acid and stained with the same solution containing 0.02% Coomassie G-250; destaining was obtained by shaking the gels for 48 hr in 10 % acetic acid.

RNA isolation

Total RNA was extracted from livers following Chomczynski and Sacchi [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thyocyanatephenol-chloroform extraction[END_REF]. The yield and quality of RNA were assessed by the 260/280 nm optical density ratio and by electrophoresis under non-denaturing conditions on 1.8% agarose gel. Ambion's DNA-freeTM kit (Ambion Europe Ltd., UK) was used to remove contaminating DNA from RNA preparations. Then, 2.0 μg of total RNAs in 20 μl total volume were retro-transcribed to obtain cDNA using Superscript II Reverse

Real time quantitative PCR

Real Time PCR reactions were performed on the DNA Engine Opticon 2 System (MJ Research, Boston, MA) in 20 l total volume with 4 l of the cDNA sample, obtained diluting (1:30) cDNA preparation, and 0.3 M of each primer using the DyNAmo™ HS SYBR® Green qPCR Kit (Finnzymes, Espoo, Finland), according to the manufacturer's instructions. Primers (PRIMM Biotech Products and Services, Milan, Italy) used for the amplification, designed using Primer3 software [START_REF] Rozen | Primer3 on the WWW for general users and for biologist programmers[END_REF], were: NRF-1 forward, 5'-aaattgggccacattacaggg-3'; NRF-1 reverse, 5'-gttgcatctcctgagaagcg-3'; NRF-2 α1 forward, 5'gggaggtggatgtaatgtgg-3'; NRF-2 α1 reverse, 5'-tgggcctggaactacaactc-3'; PGC-1forward, 5'cgcagagagtatgagaagcg-3'; PGC-1 reverse, 5'-aagcgtcacaggtgtaacgg-3'; -actin forward, 5'gccaaccgtgaaaagatgac-3'; -actin reverse, 5'-agcgcgtaaccctcatagat-3'. Data normalization was performed using β-actin as housekeeping gene. The amplification protocol was as follows: 1 cycle of 15 min at 95 °C, 39 cycles of 95 °C for 15 s, 56 °C (annealing for PGC-1 gene primers), 58 °C (annealing for NRF-1 gene primers), and 64.0 °C (annealing for NRF-2 gene primers), for 20 s, 72 °C for 20 s, plus an extension at 72 °C for 5 min.

Experiments were carried out in duplicates or triplicates. The relative expression value of treated rats with respect to hypothyroid rat signal value was calculated as fold change with the formula 2 -ΔΔCt . For each value four independent experiments were performed.

.Western blotting

Liver fragments were gently homogenized (1:10, w/v) in 500 mM NaCl, 0.5% nonidet P-40, 6 mM EDTA, 6 mM EGTA, 1 mM dithiotreitol, 40 mM Tris-HCl, pH 8.0, in the presence of antiprotease mixture including 40 μg/ml PMSF, 5 μg/ml leupeptin, 5 g/ml aprotinin, 7 g/ml pepstatin. Homogenates were centrifuged at 1000 g for 10 min at 4°C and resulting supernatants were electrophoresed through 6% stacking and 12% running SDS-PAGE gel as previously described for mitochondrial proteins. Separated hepatic A c c e p t e d M a n u s c r i p t 9 proteins were transferred to nitrocellulose membranes by electroblotting. Membranes were incubated with a 1:1000 dilution of antibodies to PGC-1, NRF-1, and NRF-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 154 mM NaCl , 10 mM Tris-HCl, pH 8.0, 2.5% non-fat dry milk, 10% Tween 20. Rabbit polyclonal antibodies raised against amino acids 1-300 mapping near the N-terminus of PGC-1, 204-503 mapping at the C-terminus of NRF-1, and 1-180 of NRF-2α, were used. Antibody binding was detected by carrying out secondary antibody incubations using peroxidase-conjugated anti first IgG antibodies (Santa Cruz Biotechnology) diluted 1:4000. Secondary antibody was detected using the ECL system according to the manufacturer's recommendation (Santa Cruz Biotechnology). The blots were stripped by treating them for 10 min with 0.2 M NaOH followed by 5-min wash with H 2 O and two 5-min washes with 154 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.1% Tween 20. The blots were again blocked for 30 min 154 mM NaCl, 10 mM Tris-HCl, pH 8.0, 2.5% non-fat dry milk, 10% Tween 20T, washed as above, and incubated for 2 h with a 1:2000 dilution of anti-actin antibody (Santa Cruz Biotechnology) in blocking solution. Remaining procedures, as described for other antibodies, were followed. The actin was used for loading standardization.

To compare protein expression levels among groups, a standard hypothyroid sample was run on each gel and all group values were then compared with the hypothyroid sample that was assigned a value of 1.

Data analysis

The data, expressed as means standard error, were analyzed with a one-way analysis of variance method.

When a significant F ratio was found, the Student-Newman-Keuls multiple range test was used to determine the statistical significance between means. Probability values (P) < 0.05 were considered significant. In Fig. 2 the results of the experiments are presented as sample curves.

Results

Changes in thyroid state were documented by modifications in heart weight/body weight ratio, and plasma levels of FT 3 and FT 4 . The body weights, which were 24310, 23910, 2397, and 2576 g for H, HT 4 , HT 3 , and HT 3 +CE rats, respectively, were not significantly affected (P>0.05) by cold exposure and hormonal treatment. Conversely, the heart weight increased differently in hyperthyroid rats so that such A c c e p t e d M a n u s c r i p t 10 animals exhibited a heart/body weight ratio increased in comparison to the controls (2.170.04). The ratio was significantly lower (P<0.05) in T 4 -treated (2.500.04) than in both groups of T 3 -treated rats (3.180.17 and 3.100.06 for HT 3 and HT 3 +CE rats, respectively). As shown in Fig. 1, plasma levels of FT 3 were higher in both groups of T 3 -treated rats, whereas FT 4 levels were higher in T 4 -treated rats.

Oxygen consumption

Data on rates of both succinate-and pyruvate/malate-supported oxygen consumption by liver preparations are offered in Fig. 2. Such rates were increased by hormonal treatment in homogenates during both state 4 and state 3 respirations irrespective on substrate. The lowest rates were reached in HT 4 group, whereas the highest ones were reached in HT 3 +CE group.

During mitochondrial state 4 respiration, all rates were increased by hormonal treatment irrespective of substrate and reached the lowest and highest values in HT 4 and in HT 3 , respectively. During state 3 respiration, all rates were also increased in treatment groups. However, in the presence of succinate they reached the highest values in HT 3 group and were not different in HT 4 and HT 3 +CE groups, whereas in the presence of pyruvate/malate the rates reached the highest values in HT 3 and HT 3 +CE groups.

Activity of complexes of respiratory chain

As shown in Fig. 3, the activity of Complex I was increased by T 4 treatment and in the greater measure by T3 treatment irrespective of cold exposure. Even the activity of Complex II was differently increased by T 4 and T 3 treatment, but cold exposure of HT 3 rats made such an activity no significantly different from that of HT 4 rats. The activities of Complex III and Complex IV were increased in all treatment groups, but the highest values were obtained in HT 3 group and the lowest ones in HT 4 group.

Cytochrome oxidase activity and mitochondrial protein content

As shown in Fig. 4, hormonal treatments were associated with increases in COX activities in homogenates, which were lower in HT 4 than in HT 3 rats. The cold exposure of T 3 -treated rats was associated A c c e p t e d M a n u s c r i p t 11 with further increase of homogenate COX activity. Liver mitochondrial protein content was not modified by hormonal treatment, but was increased when T 3 treatment was associated with cold exposure.

Mitochondrial protein analysis

The results concerning SDS-PAGE analysis of mitochondrial proteins are reported in Fig. 5. It is apparent that the treatments differentially affect the mitochondrial protein profile. In particular, the profile showed by HT 4 and HT 3 +CE groups differs from that showed by HT 3 group in a band around approximately 95 kDa and in a band above 116 kDa.

mRNA expression

As the m-RNA expression levels were too low for suitable northern blot analyses (data not shown), we performed quantitative Real Time PCR. As shown in Fig. 6, PGC-1 expression levels were increased in all treatment groups. The increases were moderate in T 4 -and T 3 -treated rats (3.5-fold) and more high (9-fold) in T 3 -treated and cold exposed rats. Different pattern was obtained for NRF-1 and NRF-2 expression levels. Indeed, NRF-1 expression reached higher levels only in T 3 treated rats and was restored to hypothyroid levels after cold exposure. Levels of NRF-2 expression were decreased by treatments, but they were lower in HT 4 than in HT 3 and HT 3 +CE rats.

Protein expression

In order to verify whether modified concentrations of mRNA molecules also lead to similar directional changes in protein abundance, we performed an analysis by western blot. Western blot experiments were conducted using identical amounts of total protein extract from livers of hypothyroid and treated rats that were loaded onto an SDS-PAGE, and blotted according to standard protocols. The high specificity of the antibodies and the molecular weight markers allowed us to easily identify the NRF-1, and NRF-2 proteins.

The specificity of the PGC-1 antibody was relatively lower, but the protein was clearly identified by its molecular weight.

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

The results reported in Fig. 7 show that PGC-1, NRF-1, and NRF-2 levels were increased by T 4 treatment and in greater measure by T 3 treatments. Cold exposure of T 3 -treated rats produced further increase in PGC-1 and NRF-2 levels, so that they were significantly higher than those found in HT 4 and HT 3 rats. Conversely, NRF-1 levels were reduced to values not significantly different than those found in HT 4 group.

We wanted to establish whether there is a relationship between protein levels and COX activities in treatment groups. Thus we found that treatment-evoked changes in PGC-1 and NRF-2 protein expression were matched by parallel changes in COX activity Conversely, no relationship was found between NRF-1 protein expression and COX activity (Fig. 8).

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

Discussion

The iodothyronine role in the liver response to cold was previously investigated comparing such a response with those elicited by euthyroid rat treatments with T 3 or T 4 (Venditi et al., 2006a). The present protocol allows to compare metabolic characteristics of rats whose serum substantially contains either T 4 or T 3 . It also allows to investigate the possible synergic effects of T 3 and catecholamines, because, plasma noradrenaline levels, which are low or normal in hyperthyroidism [START_REF] Stoffer | Plasma catecholamines in hypothyroidism and hyperthyroidism[END_REF], remarkably increase during cold exposure [START_REF] Storm | Thyroid hormone-catecholamines interrelationship during exposure to cold[END_REF][START_REF] Peralta | Modulation of mitochondrial nitric oxide synthase and energy expenditure in rats during cold acclimation[END_REF].

Thyroid hormone actions are mediated by controlling the expression of specific genes through binding to nuclear thyroid hormone receptors. The affinity of T 3 analogs for such receptors is correlated to their biological activity [START_REF] Koerner | Binding of selected iodothyronine analogues to receptor sites of isolated rat hepatic nuclei[END_REF], so that observation that T 4 and T 3 differentially increase aerobic capacity of liver preparations agrees with the lower affinity of T 4 for hepatic nuclear sites [START_REF] Koerner | Binding of selected iodothyronine analogues to receptor sites of isolated rat hepatic nuclei[END_REF]. We also found that cold exposure of T 3 -treated rats further increases homogenate aerobic capacity, whereas decreases the mitochondrial one. The cold-linked changes were associated with enhancement in tissue content of mitochondrial proteins which was not modified by hormonal treatment. This result is consistent with previous observations that T 3 does not modify number [START_REF] Goglia | The effect of thyroid state on respiratories activities of three rat liver mitochondrial fractions[END_REF] and protein mass (Venditti et al., 2006a) of liver mitochondria, whereas cold induces mitochondrial proliferation [START_REF] Goglia | Thyroid state and mitochondrial population during cold exposure[END_REF][START_REF] Goglia | Alteration in hepatic mitochondrial compartment of cold-acclimated rats. Association with enhanced triiodothyronine serum levels. A morphometric/stereologic study by electron microscopy[END_REF] and increases mitochondrial protein content [START_REF] Venditti | Cold-induced hyperthyroidism produces oxidative damage in rat tissues and increases susceptibility to oxidants[END_REF]Venditti et al., 2006a). On the other hand, our results do not support the previous hypothesis (Venditti et al., 2006a) that thyroxine is a factor responsible for cold-linked mitochondrial proliferation. Indeed, T 4 treatment does not increase mitochondrial proteins that, conversely, are increased by cold exposure of T 3 -treated rats in which T 4 plasma levels are low.

The modifications induced by treatments in the in vitro State 3 oxygen consumption by liver preparations were consistent enough with the changes in aerobic capacities. However, cold exposure of T 3 -treated rats lowered mitochondrial COX activity of about 8%, decreased succinate-sustained mitochondrial respiration in greater measure (about 24%) and did not modify that sustained by pyruvate/malate. These results are consistent with observation that cold exposure of euthyroid rats increases liver mitochondrial respiration less than T 3 treatment, although the serum FT 3 levels are not different, and cold-induced increases are different in the presence of succinate (10%) and pyruvate/malate (45%) (Venditti et al., 2006b). Our previous and A c c e p t e d M a n u s c r i p t present data suggest that cold exposure differentially affects respiratory chain components. This idea is supported by finding that cold exposure of T 3 -treated rats decreases the activities of all multi-subunit complexes other than Complex I. In the light of the enhanced mitochondrial protein content, the above finding also indicates that hepatic levels of Complex I components increase following cold exposure. The ability of cold exposure to differentially regulate mitochondrial components was also shown by SDS-PAGE analysis of mitochondrial proteins, which, in particular, confirmed previous report that cold exposure, but not T 3 , increases the concentration of a component with molecular weight higher than 116 kDa (Venditti et al., 2006b). These results indicate that in cold-exposed rats factors are operative which are involved in proliferation of mitochondria and differential induction of respiratory chain components. On the other hand, it seems that T 3 , although does not induce mitochondrial proliferation, is able to increase the mitochondrial concentration of respiratory chain components in agreement with early observation that thyroid hormone administration selectively increases the area of the mitochondrial inner membranes (Jakovcic et al., 1978). It is, however, possible that mitochondrial proliferation requests the concomitant action of more than one factor and T 3 contributes to phenomenon in conditions, such as cold exposure, in which additional factor is present.

Several reports support the idea that such an additional factor can be the noradrenaline.

The expression of respiratory apparatus is controlled by nuclear regulatory proteins including nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2), and the peroxisomal proliferator-activated receptor-γ coactivator (PGC-1). NRF-1 and NRF-2 are transcriptional factors which have been linked to the transcriptional control of many genes involved in mitochondrial function and biogenesis [START_REF] Scarpulla | Nuclear activators and coactivators in mammalian mitochondrial biogenesis[END_REF].

PGC-1 is a transcriptional coactivator which appears to play a role as an intermediary between environmental stimuli and transcriptional responses [START_REF] Scarpulla | Nuclear activators and coactivators in mammalian mitochondrial biogenesis[END_REF].

It has been shown that T 3 triggers processes, such as mitochondrial biogenesis, adaptive thermogenesis and hepatic gluconeogenesis [START_REF] Yen | Physiological and molecular basis of thyroid hormone action[END_REF], which resemble those regulated by PGC-1, which, in turn, interacts with several nuclear hormone receptors including thyroid hormone receptor β [START_REF] Puigserver | A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis[END_REF]. It has also been reported that liver PGC-1 and NRF-1 mRNA expression levels increase 6 h after T 3 treatment of hypothyroid rats and remains high for different periods (42 and 18 h, respectively). Conversely, NRF-2 expression levels do not change within the first 24 h and decrease after 48 h [START_REF] Weitzel | Two thyroid hormone-mediated gene expression patterns in vivo identified by cDNA expression arrays in rat[END_REF]. Subsequently, T 3 treatment has been found to increase liver PGC-1 protein level in measure lower than A c c e p t e d M a n u s c r i p t mRNA level [START_REF] Weitzel | Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats[END_REF]. Increases of different extent in PGC-1 mRNA and protein levels (4.5 and 2.3 fold, respectively) have also been found 24 h after T 3 addition to hepatocytes [START_REF] Zhang | Peroxisomal proliferators activated receptor-γ coactivator-1α (PGC-1α) enhances the thyroid hormone induction of carnitine palmitoyltransferase I (CPT-Iα)[END_REF].

Significant increases in PGC-1 protein levels after T 3 treatment (5 days) have been found in a variety of tissues, including liver [START_REF] Irrcher | PPARgamma coactivator-1alpha expression during thyroid hormone-and contractile activity-induced mitochondrial adaptations[END_REF]. Although recent report indicates that thyroid hormone-mediated gene expression patterns are not completely depending on PGC-1 activation [START_REF] Wulf | T 3 -mediated gene expression is independent of PGC-1alpha[END_REF], the above data support the idea that a thyroid hormone-mediated activation of PGC-1 might help this coactivator to exert its function in adaptation to endocrine signals. On the other hand, additional factors might contribute to PGC-1 activation in response to other signals, such as low environmental temperature. Indeed, PGC-1 mRNA is induced in the mouse brown fat by cold and β-adrenergic agonist treatment which mimics the coldinduced sympathetic innervation of brown fat [START_REF] Puigserver | A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis[END_REF], supporting the view that PGC-1 is a transcriptional coactivator of adaptive thermogenesis. To illustrate how PGC-1 can link the external environment to mitochondrial biogenesis and gene expression a model has been proposed in which noradrenaline, released following cold exposure, leads to PGC-1 expression induction through elevation of cellular cAMP [START_REF] Wu | Mechanisms controlling mitochondrial biogenesis and function through the thermogenic coactivator PGC-1[END_REF].

In the light of the aforementioned results, we measured mRNA levels of PGC-1, NRF-1, and NRF-2 to better understand transcript levels and their regulation on cold-induced changes in rat liver mitochondrial population. Thus, we found that only PGC-1 levels are related to cytochrome oxidase activity, respiratory characteristics, and mitochondrial protein content in liver homogenates. However, investigations of the steady-state level of mRNA concentrations are hampered by the question of whether modulation of the mRNA concentration also reflects a modulation of the protein concentration. Hence, we also tested PGC-1, NRF-1, and NRF-2 protein expression levels and found patterns of protein expression different from those of mRNA expression. PGC-1 protein expression pattern was similar enough to that of mRNA expression, but the cold-induced increase in protein levels was much lower than that in mRNA levels. Moreover, NRF-1 proteins levels reached the highest value in T 3 -treated group, but levels in T 4 -treated and cold-exposed rats were significantly higher than controls. Finally, the pattern of NRF-2 protein was completely different from that of mRNA, because protein levels in all treatment groups were higher than controls and the highest values were found in cold exposed rats. We are not able to explain the differences found in treatment effects on levels of NRF-1 mRNA and protein expression. Conversely, the results concerning NRF-2 levels are A c c e p t e d M a n u s c r i p t 16 consistent with the reported lack of a correlation between NRF-2 mRNA and protein levels in rat tissues, which has suggested that NRF-2 expression may be regulated by a posttranscriptional mechanism [START_REF] Vallejo | Evidence of tissue-specific, post-transcriptional regulation of NRF-2 expression[END_REF].

The changes in protein expression levels appear to match better with the treatment-linked metabolic modifications. In fact, our data show that hormonal treatment induces PGC-1 protein expression, which occurs coincident with increases in transcription factor expression and oxidative capacity. However, they are not able to supply an exhaustive explanation of the changes induced by treatments in characteristics of mitochondrial population. In agreement with previous observations [START_REF] Irrcher | PPARgamma coactivator-1alpha expression during thyroid hormone-and contractile activity-induced mitochondrial adaptations[END_REF], we found that the changes induced by treatments in PGC-1 protein expression are largely matched by parallel changes in COX activity. Similar pattern was also found for NRF-2 protein expression. Because thyroid hormone increases expression of mitochondrial and nuclear encoded subunits of COX in rat liver [START_REF] Sheehan | Tissue specific regulation of cytochrome c oxidase subunit expression by thyroid hormone[END_REF], it is conceivable that PGC-1 and NRF-2 protein expression also correlates with COX protein concentration.

Conversely, no relationship was found between NRF-1 levels and COX activities, because of the unexpected fall in NRF-1 levels following cold exposure of T 3 -treated rats. This result disagrees with the previous observation that biogenesis of muscle mitochondria stimulated by PGC-1 requires the function of NRF-1 [START_REF] Wu | Mechanisms controlling mitochondrial biogenesis and function through the thermogenic coactivator PGC-1[END_REF]. On the other hand, the finding that cold-induced increase in COX activity in zebrafish muscle is associated with increased NRF-1 mRNA levels, but unmodified PGC-1 mRNA levels (McClelland e al., 2006) suggests that the regulatory proteins and the mechanisms involved in mitochondrial biogenesis can be species and tissue-dependent.

In conclusion, our data supply strong indication that PGC-1 and NRFs are responsible for the increases, induced by hormonal treatment, in respiratory chain components. Conversely, other investigation needs to clarify their possible role in cold-induced mitochondrial proliferation in rat liver.

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

Figure 1. Serum levels of free triiodothyronine (FT 3 ) and thyroxine (FT 4 ) in hypothyroid (H), hypothyroid T 4treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats.

Values are means  S.E.M. For each value eight rats were used. a significant vs. H rats; b significant vs. HT 4 rats. The level of significance was chosen as P < 0.05.

Fig. 2.

Rates of oxygen consumption by liver homogenates and mitochondria from hypothyroid (H), hypothyroid T 4 -treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats.

Values are means  S.E.M. For each value eight rats were used. a significant vs. H rats; b significant vs. HT 4 rats; c significant vs. HT 3 rats. The level of significance was chosen as P < 0.05. Fig. 3. Activities of mitochondrial respiratory complexes in liver from hypothyroid (H), hypothyroid T 4 -treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats.

Values are means  S.E.M. For each value eight rats were used. a significant vs. H rats; b significant vs. HT 4 rats; c significant vs. HT 3 rats. The level of significance was chosen as P < 0.05. Values are means  S.E.M. For each value eight rats were used. a significant vs. H rats; b significant vs. HT 4 rats; c significant vs. HT 3 rats. The level of significance was chosen as P < 0.05.

  Invitrogen, San Giuliano Milanese, Italy), following manufacturer's instructions. cDNA preparation was used to perform Real Time PCR analysis.

Fig. 4 .

 4 Fig. 4. Cytochrome oxidase (COX) activity and mitochondrial protein content in liver from hypothyroid (H), hypothyroid T 4 -treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats.

Fig. 5 .

 5 Fig.5. Electrophoretic analysis of liver mitochondria. from hypothyroid (H), hypothyroid T 4 -treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats. The mitochondrial preparations (1 mg protein/ml) were solubilized with (2%) sodium dodecyl sulfate and (10%) mercaptoethanol, subjected to electrophoresis in 12% polyacrylamide gels, and stained with Coomassie blue as described in

  . Molecular weight markers are shown on the left of protein stain panel. The blots shown are representative of five independent analyses for each group.

Fig. 6 .

 6 Fig.6. Levels of PGC-1, NRF-1, and NRF-2 mRNA expression in liver from hypothyroid (H), hypothyroid T 4treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats, were analysed by quantitative Real Time PCR.

Fig. 7 .

 7 Fig.7. Levels of PGC-1, NRF-1, and NRF-2 protein expression in rat liver. Liver total proteins from hypothyroid (H), hypothyroid T 4 -treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats, were isolated and analysed using Western blot analyses. A representative result of 3 independent experiments is shown. Values reported in graphs are means  SEM of three independent

Fig. 8 .

 8 Fig. 8. Relationship between protein expression and COX activity in liver from hypothyroid (H), hypothyroid T 4 -treated (HT 4 ), hypothyroid T 3 -treated (HT 3 ), and hypothyroid T 3 -treated and cold-exposed (HT 3 +CE) rats. Values of COX activity in treatment groups were expressed relative to values found in hypothyroid group.

  Fig. 1

Fig. 2

 2 Fig. 2

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  Fig. 6

  Fig. 7

Acknowledgements

This work was supported by grants from the Italian Ministry of University and Scientific and Technological Research.


Martin Blixt
email: martin.blixt@mcb.uu.se

Bo Niklasson

Stellan Sandler

Suppression of bank vole pancreatic islet function by proinflammatory cytokines

Keywords: proinflammatory cytokines, interleukin -1, tumor necrosis factor -, interferon -, pancreatic islets, bank vole

Summary

We recently characterized -cell function of pancreatic islets from normal and glucose intolerant/diabetic bank voles. These animals had features of both human type 1 and type 2 diabetes. Presently, we studied how pancreatic islets isolated from normal i.e. glucose tolerant bank voles are affected by proinflammatory cytokines in vitro.

IL-1 alone or in combination with TNF- + IFN- markedly reduced insulin gene expression and the (pro)insulin biosynthesis rate, which was accompanied by a profound depletion of the islet insulin content. The decrease of islet insulin content was not prevented by the preferential inducible NO synthase inhibitor aminoguanidine.

Our findings suggest that the reduction in islet insulin content is not attributed to enhanced exocytosis or related to altered glucose metabolism, but rather are due to a decline in insulin production. The suppressive effects of islet functions elicited by cytokines seem to be mediated by an NO-independent mechanism. In relation to previous studies on cytokine effects on islets from various species, the bank vole islets show a pattern which more resembles human islets than rat or murine islets.

A c c e p t e d M a n u s c r i p t 3 Abstract Bank voles kept in captivity may develop diabetes. We recently characterized -cell function of pancreatic islets from normal and glucose intolerant/diabetic bank voles.

These animals had features of both human type 1 and type 2 diabetes. Cytokines may impair -cell function in both types of diabetes. Presently, we studied how pancreatic islets isolated from normal i.e. glucose tolerant bank voles are affected by proinflammatory cytokines in vitro.

Islets were exposed to hIL-1 (25 U/ml) alone or in combination with hTNF- (1000 U/ml) + mIFN- (1000 U/ml) for 48 hours, whereupon islet functions were assessed.

Cytokines markedly reduced insulin gene expression and the (pro)insulin biosynthesis rate, which was accompanied by a profound depletion of the islet insulin content. The cytokines did not affect the culture medium insulin accumulation and the glucose oxidation rate, but caused a modest increase in medium nitrite, an indicator of nitric oxide (NO) generation. Cytokine-induced decrease of islet insulin content was not prevented by the preferential inducible NO synthase inhibitor aminoguanidine.

These findings suggest that the reduction in islet insulin content is not attributed to enhanced exocytosis or related to altered glucose metabolism, but rather are due to a decline in insulin production. The suppressive effects of islet functions elicited by cytokines seem to be mediated by an NO-independent mechanism. In relation to previous studies on cytokine effects on islets from various species, the bank vole islets show a pattern which more resembles human islets than rat or murine islets.

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

Introduction

Bank voles kept in captivity develop polydipsia and polyuria and subsequently diabetes to a significant extent [START_REF] Blixt | Characterization of beta-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus[END_REF][START_REF] Schoenecker | Development of stereotypies and polydipsia in wild caught bank voles (Clethrionomys glareolus) and their laboratory-bred offspring. Is polydipsia a symptom of diabetes mellitus?[END_REF]. We have recently characterized -cell function of pancreatic islets isolated from normal and glucose intolerant/diabetic bank voles and found that this animal model may have features of both human type 1 and type 2 diabetes [START_REF] Blixt | Characterization of beta-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus[END_REF]. At least in the wild state a novel picornavirus (Ljungan virus) have been found in the pancreas of the bank voles [START_REF] Niklasson | A new picornavirus isolated from bank voles (Clethrionomys glareolus)[END_REF]. Its possible role in the development of diabetes in the bank voles remain to be proven. It has been shown that the CD1 mouse offspring develop diabetes when the mother is inoculated with the Ljungan virus during gestation [START_REF] Niklasson | Prenatal viral exposure followed by adult stress produces glucose intolerance in a mouse model[END_REF].

Cytokines play a crucial role in modulation of the immune cells during immune reactions, but they are also believed to be important for the impairment of the -cell function during the autoimmune destruction in type 1 diabetes [START_REF] Rabinovitch | Cytokines and their roles in pancreatic islet beta-cell destruction and insulin-dependent diabetes mellitus[END_REF][START_REF] Sandler | Biochemical and molecular actions of interleukin-1 on pancreatic beta-cells[END_REF]. It has also been suggested that inflammatory mediators like cytokines may be involved in the development of type 2 diabetes both at the cell level and in peripheral tissues [START_REF] Donath | Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes[END_REF][START_REF] Kolb | An immune origin of type 2 diabetes?[END_REF][START_REF] Larsen | Interleukin-1-receptor antagonist in type 2 diabetes mellitus[END_REF][START_REF] Sjoholm | Inflammation and the etiology of type 2 diabetes[END_REF]. Proinflammatory cytokines like IL-1 and TNF-, secreted from activated macrophages and T-cells, are proposed to be responsible for the activation of inducible nitric oxide synthase (iNOS) [START_REF] Mandrup-Poulsen | The role of interleukin-1 in the pathogenesis of IDDM[END_REF].

Activated iNOS produce nitrogen oxide (NO) free radicals that may perturb islet function and even be toxic to the -cell and ultimately lead to cell death [START_REF] Eizirik | Interleukin-1 beta induces the expression of an isoform of nitric oxide synthase in insulin-producing cells, which is similar to that observed in activated macrophages[END_REF][START_REF] Southern | Inhibition of insulin secretion by interleukin-1 beta and tumour necrosis factor-alpha via an L-arginine-dependent nitric oxide generating mechanism[END_REF][START_REF] Welsh | Interleukin-1 beta-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase[END_REF]. In rodent islets iNOS is activated when exposed to IL-1 alone or when added in combination with TNF- or IFN- or both. However, in human islets a combination of IL-1 + IFN- or IL-1 + TNF- + IFN- are needed to activate iNOS [START_REF] Eizirik | Cytokines suppress human islet function irrespective of their effects on nitric oxide generation[END_REF].

Presently, we have studied how pancreatic islets isolated from normal i.e. glucose tolerant bank voles are affected by prolonged exposure to proinflammatory cytokines in vitro. We have chosen to use IL-1 alone and in combination with TNF- and IFN- to ascertain iNOS activation if possible. Indeed, it has been shown that pancreatic islets of various species may exhibit differences in response to these cytokines. Herein we report that bank vole islets are affected by cytokine exposure, but in contrast to rat and mouse islets treated in a similar way the functional changes seem to be NO independent.

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

Materials and methods

Animals

Bank voles (Myodes glareolus) were housed at Astrid Fagreus Laboratory, Karolinska Institute, Sweden with free access to water and standard laboratory chew.

The experimental procedures were approved by the animals' ethical committee in Stockholm (N248/03) and in accordance with international guidelines (NIH publications no. 85-23, revised 1985).

Male bank voles 15-20 weeks of age were tested with a glucose tolerance test (GTT) were 2 g glucose/kg body weight was injected IP. Blood glucose measurement was performed with an automated glucose meter (Accu-Chek Aviva; Roche Diagnostics, Bromma, Sweden) immediately prior to injection (0 minutes), at 60 and 120 minutes after glucose injection. Animals with 120 min blood glucose < 11.1 mM were classified as glucose tolerant. The pancreas in such animals was resected and placed in Hanks´ balanced salt solution (HBSS; SBL Vaccine, Stockholm, Sweden) supplemented with 50 U/ml benzylpenicillin and 0.05 mg/ml streptomycin. The animals were anaesthetized with Isoflurane (Abbott Scandinavia, Solna, Sweden) prior to testing and killing.

Islet isolation, culture and cytokine exposure

Pancreatic islets were isolated by collagenase digestion as described elsewhere [START_REF] Blixt | Characterization of beta-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus[END_REF]. Islets from individual bank voles were maintained in medium RPMI 1640 with 11.1 mM glucose supplemented with 10 % fetal calf serum, 2 mM glutamine and 50 U/ml benzylpenicillin and 0.05 mg/ml streptomycin. Islets were precultured at 37 °C, in humidified air + 5 % CO 2 for 5 days were upon the preculture islets were transferred to new culture dishes containing culture medium as above, with or without addition of IL-1 (25 U/ml corresponding to 144.5 pM; human; PeproTech EC Ltd, London, UK) alone or in combination with TNF- (1000 U/ml corresponding to 143.7 pM; human; PeproTech) + IFN- (1000 U/ml corresponding to 160.3 pM; murine; PeproTech) and cultured for 48 hours before tested. In separate experiments we also added aminoguanidine, which is a preferential inhibitor of iNOS [START_REF] Holstad | Inhibition of nitric oxide formation by aminoguanidine: an attempt to prevent insulin-dependent diabetes mellitus[END_REF], to islet cultures with or without the cytokine combination during the 48 hours culture period.

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

Medium insulin accumulation, glucose-stimulated insulin release and islet insulin content

Culture medium was collected for determination of medium insulin accumulation.

Islets in triplicate of 10 were incubated at 1.7 mM glucose in KRBH supplemented with 2 mg/ml BSA at 37 °C (O 2 /CO 2 ; 95/5 %) for 1 hour. Subsequently the buffer was replaced with KRBH supplemented 16.7 mM glucose and incubated in an additional hour. After the incubations the islets were pooled in groups of 30 and dispersed though sonication in H 2 O. The islet insulin was extracted at 4 °C in 70 % ethanol supplemented with 0.13 mM HCl. The insulin concentrations were measured with rat insulin ELISA and high range rat insulin ELISA kit (Mercodia AB, Uppsala, Sweden).

Islet total protein and (pro)insulin biosynthesis and glucose oxidation rates

Groups of 10 islets in duplicate were incubated in KRBH containing either 1.7 mM or 16.7 mM glucose and 3 H-labeled leucine (protein and (pro)insulin biosynthesis), or KRBH supplemented 16.7 mM glucose and D-[U-14 C]glucose (glucose oxidation rate) and further analysis was performed as described previously [START_REF] Blixt | Characterization of beta-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus[END_REF].

Medium nitrite

Culture medium samples were mixed with 0.05 % N-(1-naphtyl)-ethylenediamine dihydrochloride and 0.5 % sulfanilamide dissolved in phosphoric acid and incubated at 60 °C in 2 minutes. Nitrite levels were determined through absorbance measurement at 546 nm in a spectrophotometer. Culture media collected from wells without islets was used as measurement of the original nitrite level in the media.

RNA isolation, cDNA synthesis and Real Time PCR

50 islets from each group were used to extract total RNA with RNeasy Micro kit (Qiagen, Hilden, Germany) complemented with RNase-free DNAse (Qiagen) and eluted in 14 µl of RNAse free water. The RNA amount and purity was tested using the Nanodrop ND-1000 system (NanoDrop Technologies, Delaware, USA).

Synthesis of cDNA was performed with Reverse Transcription System (Promega, Madison, USA) using 1:2 volume of total RNA and 0.025 μg/µl Oligo(dT)15 primer. The reactions were incubated at 42 C in 60 minutes followed by 99 C in 5 minutes.

The LightCycler Instrument (Roche) combined with sequence independent detection with SYBR Green I was used to amplify and analyze generated cDNA. The sequence of the primers used were: insulin (mouse) forward 5´- samples with the second derivative maximum method [START_REF] Gibson | A novel method for real time quantitative RT-PCR[END_REF]Heid et al., 1996). The relative expression was calculated from the formula 2 -(Ct) .

Viability

Islet viability was determined using Hoechst 33342 and propidium iodide. In each test 10 islets were incubated with 5µg/ml bisbenzimide and 20 µg/ml propidium iodide for 20 minutes. The islets were washed in PBS and the emitted fluorescence was observed at 461 nm and 615 nm respectively after excitation with UV light. The nucleus morphology was assessed and cells were counted by a blinded observer using a fluorescence microscope.

Statistical analysis

A mean was calculated from each duplicate or triplicate groups of islets and then considered as one separate observation. Each observation represents an individual bank vole islet donor. Values are expressed as means  S.E.M., and groups of data were compared using Student's t-test, ANOVA with a Bonferroni correction or Mann-Whitney rank sum test. Tests are considered statistically significant when P  0.05. Statistical analysis was performed using SigmaStat (SPSS Inc., Chicago, IL, USA).

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

Results

Islets isolated from glucose tolerant bank voles and exposed to IL-1or a combination of IL-1 + TNF- + IFN- showed similar medium insulin accumulation as compared to non-cytokine exposed control islets (Table 1). Islet insulin secretion was affected by the proinflammatory cytokines. After culture it was seen that the basal insulin release at low glucose (1.7 mM) was elevated from islets cultured with the cytokine combination, but not to a significant extent after addition of IL-1 alone (Table 1). At high glucose (16.7 mM) there was a trend towards a decline in insulin release after cytokine addition. However, the relative stimulation of insulin secretion at high glucose vs. low glucose was lowered following addition of IL-1 alone or of the cytokine mixture. The islet insulin content was markedly decreased after culture in the presence of either IL-1or the combination of IL-1 + TNF- + IFN- (Table 1). Also the islet oxidative metabolism was assessed by determination of glucose oxidation rates at 16.7 mM glucose (Table. 1). Thus, it was found that neither IL-1 nor the combination of cytokines affected the glucose oxidation rates.

Next, we measured the islet capacity to synthesize proteins and (pro)insulin at a low and high glucose concentration (Table 2). Due to scarcity of islets, incubations at 1.7 mM glucose after culture with the cytokine combination were not performed. IL-1 did not affect the (pro)insulin or total protein biosynthesis rates at low glucose. The (pro)insulin biosynthesis rate at high glucose was impaired after culture with both IL-1 alone and the cytokine combination. The total protein biosynthesis was also reduced after addition of the cytokine mixture. The (pro)insulin biosynthesis rates were more suppressed compared to protein biosynthesis rates, which resulted in a substantial decline in the relative contribution of labeled (pro)insulin to the total pool of labeled proteins, although for the cytokine combination this was barely not statistically not significant (P = 0.06).

In subsequent experiments we assessed transcription level of the insulin and PDX-1 (Table 3). Insulin gene transcription was reduced by approximately 50 % after cytokine addition. PDX-1 was also expressed in bank vole islets and also this transcript tended to be reduced, but this difference did not attain statistical significance.

We also determined the amount of non-viable islet cells by staining with Hoechst 33342 and propidium iodide after culture with the cytokine combination. There was not an increase in death of bank vole islet, but rather on the contrary there was A c c e p t e d M a n u s c r i p t 9 tendency (P = 0.06) towards a decrease in the number of non-viable islet cells after exposure to IL-1 + TNF- + IFN- compared to control islets (Figure 1).

As an indicator of NO formation nitrite accumulation in the culture medium was measured. There was a modest increase in nitrite after addition of IL-1 alone, but no significant increase after adding the cytokine combination (Table 1). In order to examine if the NO production may be attributed to iNOS activity, aminoguanidine (2 mM) a preferential iNOS inhibitor [START_REF] Holstad | Inhibition of nitric oxide formation by aminoguanidine: an attempt to prevent insulin-dependent diabetes mellitus[END_REF] was used (Figure 2A).

Addition of aminoguanidine reduced the medium nitrite level both from noncytokine and cytokine exposed islets. However, the reduction in islet insulin content induced by the cytokine combination was not counteracted by aminoguanidine (Figure 2B), suggesting that the decline in islet insulin content was not NOdependent.

Finally, we also performed some experiments with addition of IL-1 (25 U/ml) alone for only 24 h. This resulted in a decrease in the islet insulin content (ng/10 islets; Control: 292 ± 70 and IL-1: 90.5 ± 14.6; P<0.01 (n = 6)), but no significant effects on medium insulin accumulation and glucose-stimulated insulin release was seen (data not shown).

Discussion

The aim of the present study was to investigate if proinflammatory cytokines affect the function of pancreatic islets isolated from bank voles in vitro. This was considered of interest since the bank voles develop diabetes in captivity and the animals show signs of both type 1 and type 2 diabetes, and cytokines may have a role in both types of diabetes [START_REF] Donath | Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes[END_REF][START_REF] Kolb | An immune origin of type 2 diabetes?[END_REF][START_REF] Larsen | Interleukin-1-receptor antagonist in type 2 diabetes mellitus[END_REF][START_REF] Mandrup-Poulsen | The role of interleukin-1 in the pathogenesis of IDDM[END_REF][START_REF] Rabinovitch | Cytokines and their roles in pancreatic islet beta-cell destruction and insulin-dependent diabetes mellitus[END_REF]. We used human and mouse preparations of cytokines in our experiments because bank vole specific cytokines are not available. Despite this the cytokines elicited herein various effects when islet functions were examined.

In the present study we found that the cytokines markedly reduced the insulin gene expression and the (pro)insulin biosynthesis rate and this was accompanied by a profound depletion of the islet insulin content. The cytokines also induced a slight inhibition of glucose-stimulated insulin secretion, but they did not affect the culture medium insulin accumulation and the glucose oxidation rate. This finding suggest that the reduction in islet insulin content was not attributed to an enhanced exocytosis and not related to an altered glucose metabolism, but rather was due to a decline in the formation of insulin. induced strong suppressive effects on all functions described above and it is likely that impaired glucose metabolism is a key factor [START_REF] Sandler | Inhibitory effects of interleukin 1 on insulin secretion, insulin biosynthesis, and oxidative metabolism of isolated rat pancreatic islets[END_REF][START_REF] Sandler | Studies on the mechanisms causing inhibition of insulin secretion in rat pancreatic islets exposed to human interleukin-1 beta indicate a perturbation in the mitochondrial function[END_REF][START_REF] Welsh | Interleukin-1 beta-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase[END_REF]. Concerning mouse pancreatic islets exposed to IL-1 the insulin content and (pro)insulin biosynthesis rates were depressed, medium insulin accumulation and glucose oxidation rates essentially unaffected, but glucosestimulated insulin release reduced by about 50% [START_REF] Eizirik | Interleukin-1 beta depletes insulin messenger ribonucleic acid and increases the heat shock protein hsp70 in mouse pancreatic islets without impairing the glucose metabolism[END_REF]. Furthermore, human islets cultured with IL-1 alone for a prolonged period show a stimulated cell function [START_REF] Eizirik | Predominance of stimulatory effects of interleukin-1 beta on isolated human pancreatic islets[END_REF], and when a combination of cytokines was added a strong increase in medium insulin accumulation, a decrease in islet insulin content, a decline in relative glucose-induced insulin release (16.7/1.7), but no change in glucose oxidation rate was seen [START_REF] Eizirik | Cytokines suppress human islet function irrespective of their effects on nitric oxide generation[END_REF]. When analyzing the absence and presence of functional alterations observed after prolonged treatment of bank vole islets to IL-1 or the cytokine combination, the islets of this species more resemble isolated human islets than isolated rat or murine islets.

Especially NO, via induction of the iNOS, has been found to be an important mediator of cytokine actions in -cell preparations; for a review see [START_REF] Eizirik | The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic beta cells[END_REF]. When iNOS is induced this normally leads to very marked increase in NO formation above basal NO generation formed from constitutive NOS (cNOS) enzymes. NO is mostly assessed by nitrite medium accumulation in cell culture experiments. Herein we found only a modest increase in medium nitrite after culture of bank vole islets with IL-1 and no significant increase when IL-1 + TNF- + IFN- were added. Currently, addition of aminoguanidine reduced nitrite generation from both control and cytokine exposed islets, but this effect of aminoguanidine is probably due to inhibition of cNOS activity since aminoguanidine is not a completely selective blocker of iNOS [START_REF] Holstad | Inhibition of nitric oxide formation by aminoguanidine: an attempt to prevent insulin-dependent diabetes mellitus[END_REF]. However, this effect was not accompanied by a protection against cytokine mediated decrease in islet insulin content (Figure 2B). This suggests that it is unlikely that iNOS and excessive NO production would have contributed to the inhibitory actions we observed in the present study. Thus, NO-independent suppressive actions elicited by the cytokines must be considered in this context. Indeed, such mechanisms have been suggested to occur based on experiments with human islets [START_REF] Eizirik | Cytokines suppress human islet function irrespective of their effects on nitric oxide generation[END_REF], rat-derived cell preparations [START_REF] Dunger | Tumor necrosis factor-alpha and interferon-gamma inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement[END_REF][START_REF] Suarez-Pinzon | Mechanisms of cytokine-induced destruction of rat insulinoma cells: the role of nitric oxide[END_REF] and pancreatic islets from mice with an inactivated iNOS gene [START_REF] Andersson | Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase[END_REF]. In experiments performed with the latter type of islets it has been shown that mRNA expression for the transcription factor PDX-1 was reduced by cytokines (Andersson , 2005). Presently, we observed a reduction in insulin gene expression by 50% after exposure to IL-1 + TNF- + IFN- and a tendency (P = 0.087) that PDX-1 mRNA was reduced. An unresolved issue is that it can not be excluded that the colony of bank voles examined in our study is infected by the Ljungan virus. If so and whether this would have influenced our findings when cytokines were added to the isolated pancreatic islets is unclear. In this context it is of interest that it was recently reported that BB rat model for type 1 diabetes is infected with the Ljungan virus [START_REF] Niklasson | The BioBreeding rat diabetes model is infected with Ljungan virus[END_REF].

In the present study we assessed if the combinations of cytokines elicited increased cell death, but we did not observe such an effect. This lack of effect could be due to that the period of cytokine treatment was too short and/or the concentrations of cytokines were insufficient, a species (bank vole) specific cytokine action might be required, and of course it can not be excluded that the proinflammatory cytokine combination does not induce cytotoxicity to the bank vole islet cells. If anything it could be that the particular cytokine treatment protocol may reduce bank vole islet cell death in vitro. The mechanism underlying such a putative effect is unclear but one speculation is that IFN- may interfere with cell death signals occurring in the bank vole islet cells in vitro.

Another question is if the islet function of islets isolated from bank voles that will develop diabetes, is similar to the islet function observed in the present study after cytokine addition in vitro. In our previous study on islets isolated from glucose intolerant/diabetic bank voles and examined acutely after isolation, we observed that this subpopulation of islets exhibited an enhanced function perhaps due to an enhanced functional demand on the remaining islets [START_REF] Blixt | Characterization of beta-cell function of pancreatic islets isolated from bank voles developing glucose intolerance/diabetes: an animal model showing features of both type 1 and type 2 diabetes mellitus, and a possible role of the Ljungan virus[END_REF]. In contrast, in the present study in which islets were isolated from glucose tolerant bank voles and exposed to proinflammatory cytokines showed a suppression of islet functions. However, from these combined observations it can nevertheless not be established whether or not direct actions of proinflammatory cytokines on islet function in vivo play a role during development of diabetes in bank voles.

In conclusion, pancreatic islets isolated from glucose tolerant bank voles are affected by prolonged exposure to proinflammatory cytokines. This effect seems to be essentially NO-independent, and a key cellular event appears to be a decreased level of insulin mRNA leading to a decline in insulin translation and a depletion of insulin stores in the -cells. Percent non-viable islet cells in isolated bank vole islets cultured in 48 hours with or without IL-1 (25 U/ml) + TNF- (1000 U/ml) + IFN- (1000 U/ml). Islets were stained with Hoechst 33342 and propidium iodide, and cells were counted by a blinded observer using a fluorescence microscope. The number of non-viable cells are expressed as percent of total islet cell count and presented as means  S.E.M. for n observations. Data are compared using ANOVA with a Bonferroni correction where x denotes P = 0.06 vs. control islets.

Figure 2

Islets in groups of 70 were cultured for 48 hours with or without IL-1 (25 U/ml) + TNF- (1000 U/ml) + IFN- (1000 U/ml) and in the presence and absence of aminoguanidine (2 mM). Medium nitrite concentration (A) and islet insulin content (B) was subsequently determined. Values are means  S.E.M. for n observations. Data are compared using ANOVA with a Bonferroni correction where *, ** and *** denote P  0.05, P  0.01 and P  0.001 vs. control islets (no addition) x and xx denote P  0.05 and P  0.01 vs. cytokine exposed islets. The relative islet insulin and PDX-1 gene expression compared to beta actin gene expression calculated from the difference in the Ct values. The islets used were isolated from glucose tolerant bank voles and precultured in 5 days in RPMI 1640 + 10% fetal calf serum, then the islets were further cultured in fresh medium for 48 hours with 25 U/ml IL-1, 1000 U/ml TNF- + 1000 U/ml IFN- as indicated. Values are means  S.E.M. for the number of observation stated in parenthesis. Data were compared using Mann-Whitney rank sum test where * and x denotes P  0.05 and P = 0.087 vs. control islets incubated at the same glucose concentrations.

  A c c e p t e d M a n u s c r i p t 7 CCATCAGCAAGCAGGTTAT-3´, reverse 5´-GGGTGTGTAGAAGAAGCCA-3´, PDX-1 (mouse) forward 5´-GGTGCCAGAGTTCAGCGCTA -3´ reverse 5´-TTGTTTTCCTCGGGTTCCGC-3´ and -actin (mouse) forward 5´-CCACCGATCCACACAGAGTACTTG-3´ reverse 5´-GCTCTGGCTCCTAGCACC-3´ (TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) PCR amplification of 0.1 µg of cDNA sample were performed with 0.2 µM of each insulin or PDX-1 primer and 0.5 µM of each -actin primer in SYBR Green JumpStart ready mixture (FastStart Taq DNA polymerase, dNTP mix and SYBR Green I dye; Roche) and 3.5 mmol/l MgCl 2 . After an initial 95 C in 30 seconds the following was cycled 40 times; 94 C in 5 seconds, 47 C in 10 seconds and 72 C in 15 seconds. Cycle threshold (Ct) values were obtained for individual

  partly similar and partly it differs to what have been seen in experiments using islet preparations from other species. In rat pancreatic islets IL-1

  Figure 1.

  Figure1

Figure2B

  Figure2B

Table 1 .

 1 Effects of proinflammatory cytokines on insulin secretion, glucose metabolism and nitrite production of pancreatic islets isolated from bank voles.Islets from glucose tolerant bank voles were isolated and precultured for 5 days in medium RPMI 1640 + 10% fetal calf serum, then the islets were further cultured in fresh medium for 48 hours without or with addition of 25 U/ml IL-1 or in combination with 1000 U/ml TNF- + 1000 U/ml IFN- as indicated. Values are means  S.E.M. for the number observations given in parenthesis. *, ** and *** denote P<0.05, P<0.01 and P<0.001 vs. control islets, using Student's unpaired t-test.

		Control	IL-1b	IL-1b + TNF-a + IFN-g
	Medium insulin accumulation (ng/10 islets x 24 h)	481 ± 57.7 (20)	548 ± 90.1 (10)	404 ± 34.6 (14)
	Islet insulin content (ng/10 islets)	297 ± 27.0 (8)	81.1 ± 13.5 (6) *** 91.9 ± 6.8 (8) ***
	Islet insulin release
	(ng/10 islets x 1 h)
	1.7 mM glucose	18.0 ± 1.4 (8)	24.1 ± 2.6 (6)	25.8 ± 1.7 (8) *
	16.7 mM glucose	57.8 ± 5.1 (8)	38.8 ± 6.0 (6)	46.6 ± 3.6 (8)
	16.7/1.7 (fold increase)	3.6 ± 0.5 (8)	1.7 ± 0.3 (6) **	2.5 ± 0.2 (8) **
	Islet glucose oxidation rate (pmol/10 islets x 60 min)	492 ± 80.6 (13)	387 ± 56.5 (9)	395 ± 64.5 (8)
	Medium nitrite concentration (nM/10 islets)	52.8 ± 6.7 (19)	93.6 ± 16.7 (10) *	72.6 ± 7.2 (15)

Table 3 .

 3 Islet insulin and PDX-1 gene expression after 48 hours exposure to IL-1 + TNF- + INF-.

	mRNA (2 -(DCt) )	Control		IL-1b + TNF-a + IFN-g
	Insulin	42.58 ± 8.51	(8)	22.46 ± 2.68	(9) *
	PDX-1	0.0038 ± 0.0016 (6)	0.0016 ± 0.0003 (9) x

Acknowledgements

We are grateful for the excellent technical assistance by Ing-Britt Hallgren. This study was supported by grants from the Swedish Research Council (72X-8273), the Swedish Diabetes Association, the Novo Nordic Foundation and the Ernfors Fund.

A c c e p t e d M a n u s c r i p t Islet total biosynthesis and (pro)insulin synthesis rate at low (1.7 mM) glucose and high (16.7 mM) glucose conditions. Islets isolated from glucose tolerant bank voles and precultured in 5 days in RPMI 1640 + 10% fetal calf serum, then the islets were further cultured in fresh medium for 48 hours with or without addition of 25 U/ml IL-1 alone or in combination with 1000 U/ml TNF- + 1000 U/ml IFN- as indicated. Values are means  S.E.M. for the number of observation stated in parenthesis. Data are compared using ANOVA with a Bonferroni correction where *, **, *** and x denote P  0.05, P  0.01, P  0.001 and P = 0.06 vs. control islets incubated at the same glucose concentrations.


Fiorella Miceli

Franco O Ranelletti

Enrica Martinelli

Marco Petrillo

Giovanni Scambia

Pierluigi Navarra
email: pnavarra@rm.unicatt.it

Gabriella Ferrandina

Expression and subcellular localization of CRH and its Receptors in Human Endometrial Cancer

Keywords: Corticotropin-Releasing Hormone, CRH-receptors type 1 and type 2, Human Endometrial Carcinoma, Estrogen Receptor, Progesterone Receptor

CRH and its receptors are expressed in human normal endometrial cells, where they are associated to anti-proliferative progesterone-like activity. We aimed to investigate CRH, CRH-R1 and CRH-R2 expression and intracellular localization in human endometrial cancers and their relationships with tumor biological parameters. Surgical specimens were obtained from 51 untreated endometrial cancer patients and immunohistochemistry for CRH, CRH receptors, ER, PR and Ki-67 was performed. We found a diffuse cytoplasmic staining in 100%, 92 % and 60.7 % of tumor specimens for CRH, CRH-R1 and R2, respectively. At variance with tumor tissues, the surrounding normal endometrial glands exhibit a typical paranuclear/apical pattern for CRH and stained for CRH-R2 at the nuclear level, whereas CRH-R1 staining was similar to that observed in tumor area. Positive correlations were found between CRH-R1 and PR expression, as well as between CRH-R2 cytoplasmic pattern and more advanced FIGO stage disease, respectively.

Introduction

Endometrial adenocarcinoma is currently the most frequent malignancy of the female genital tract in developed countries and represents the seventh leading cause of death for cancer in women [START_REF] Jemal | Cancer statistics[END_REF]. At present, FIGO stage, depth of myometrial invasion and grade of differentiation are commonly used to predict clinical outcome and plan treatment modalities in endometrial cancer patients [START_REF] Hoskins | Principles and Practice of Gynecologic Oncology[END_REF]. However, epidemiologic observation shows 10% of stage I and 17% of stage II patients experience recurrence of disease (Petterson 1994), thus suggesting that, apart from the above mentioned parameters, biological markers more strictly related to tumor cell biology might provide additional useful information in order to improve patients' prognostic characterization and possibly individualize treatments.

In this context, attention has been focused on corticotropin-releasing hormone (CRH), a 41amino acid peptide originally characterized as a hypothalamic factor mediating behavioral, autonomic and neuroendocrine responses to stressors. The biological actions of CRH and related peptides (urocortins 1-3) are mediated through the activation of two CRH receptor isoforms, namely CRHreceptor type 1 (CRH-R1) and type 2 (CRH-R2), each exhibiting distinct functions, tissue-specific expression patterns and binding affinities toward CRH and other endogenous ligands [START_REF] Hillhouse | The molecular mechanisms underlying the regulation of the biological activity of corticotropin-releasing hormone receptors: implications for physiology and pathophysiology[END_REF]. CRH-R1 and its endogenous ligands, CRH and urocortins, were also localized in human normal endometrial tissues, where the peptide was found to exert anti-proliferative, progesterone-like effects [START_REF] Ferrari | Corticotropin releasing factor decidualizes human endometrial stromal cells in vitro. Interaction with progestin[END_REF][START_REF] Di Blasio | Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells[END_REF][START_REF] Florio | Identification of urocortin mRNA and peptide in the human endometrium[END_REF][START_REF] Florio | Urocortin expression is down-regulated in human endometrial carcinoma[END_REF]. Evidence from our group shows that similar actions are also exerted by CRH on a human endometrial tumor cell line, Ishikawa cells (IK), by the activation of CRH-R1 subtype [START_REF] Graziani | CRH inhibits cell growth of human endometrial adenocarcinoma cells via CRH-receptor 1mediated activation of cAMP-PKA pathway[END_REF][START_REF] Graziani | Corticotropin-releasing hormone receptor-1 in human endometrial cancer[END_REF].

Apart from endometrial cancer, CRH has been shown to possess growth inhibitory effects in the vast majority of tumor cells and tissues [START_REF] Melzig | Corticotropin releasing factor inhibits proliferation of AtT-20 cells[END_REF][START_REF] Graziani | Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype[END_REF][START_REF] Minas | Intratumoral CRH modulates immuno-escape of ovarian cancer cell through FasL regulation[END_REF], through the activation of CRH-R1 subtype [START_REF] Graziani | CRH inhibits cell growth of human endometrial adenocarcinoma cells via CRH-receptor 1mediated activation of cAMP-PKA pathway[END_REF][START_REF] Reubi | Expression of CRF1 and CRF2 receptors in human cancers[END_REF][START_REF] Graziani | Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype[END_REF]. Putative anti-tumor effects of CRH also appear to be mediated by CRH-R2, via an indirect mechanism involving the down-regulation of vascular endothelial growth factor (VEGF), and the subsequent inhibition of tumor vascularization [START_REF] Bale | Corticotropin-releasing factor receptor 2 is a tonic suppressor of vascularization[END_REF].

In this study, we used immunohistochemistry to investigate the expression and intracellular localization of CRH and its two receptor subtypes in endometrial cancers as well as normal surrounding tissues, in a single institutional series of primary untreated endometrial cancer patients. Correlation among morphological findings, clinico-pathological features and clinical outcome was evaluated. In particular, the reciprocal relationships among CRH, its receptors, and biological parameters such as estrogen receptor (ER), progesterone receptor (PR), and the proliferation marker Ki-67 have been analyzed.

Subjects

This study was conducted on 51 primary untreated endometrial cancer patients admitted, treated and followed-up at the Gynecologic Oncology Unit, Catholic University of Rome. In our Institution a written informed consent is routinely requested to patients for collection of their clinical data, as well as paraffin embedded sections for research use. Staging was performed according to FIGO classification (1988). Median age was 61 years (range 29-85). Forty (78.4%) patients were stage I-II, and 11 (21.6%) patients were stage III-IV disease. Most of the tumors (88.2%) were endometrioid adenocarcinomas and varied from well (23.5%) through moderate (49.0%) to undifferentiated tumors (27.5%). The other clinico-pathological characteristics are listed in Table 1.

All patients were submitted to total abdominal or modified radical hysterectomy plus bilateral salpingo-oophorectomy. At time of laparotomy fluid samples were obtained for cytologic examination and thorough intra-abdominal and pelvic exploration with biopsy or removal of any suspicious lesion was performed. Complete cytoreduction was achieved in advanced cases. Systematic pelvic and paraaortic lymphadenectomy was performed in 40 (78.4%) high risk patients (deep myometrial invasion, cervical involvement, special histotypes) according to a national randomized clinical protocol undergoing in our Department; lymph node sampling was performed in 6 cases because of visible or palpable enlarged lymph nodes. High risk patients underwent external radiotherapy or 4 to 6 cycles of cisplatin-based (each cycle: 50-75 mg/m 2 ) chemotherapy 2-3 weeks after primary surgery. After completing treatment, patients were followed by gynecological examination, transvaginal/ abdominopelvic ultrasonography, and radiological investigations, if necessary, every 4 months for the first two years, every 6 months from the third up to the fifth year and yearly thereafter.

Immunohistochemistry

Immunohistochemistry for CRH, CRH-R1 and CRH-R2 expression was performed on 3 m paraffin tissue sections mounted on poly-l-lysine-coated slides and dried at 37 °C overnight. After the slides were deparaffinized in xilene and rehydrated conventionally, the endogenous peroxidase was blocked with 0.3% H 2 O 2 in methanol for 10 min. and subjected to heat-induced epitope retrieval in microwave oven using the DAKO ChemMate detection Kit (DAKO, Glostrup, Denmark), according to the manufacturer's instructions. Endogenous biotin was saturated by a biotin blocking kit (Vector Laboratories, Burlingame CA, USA).

Cells expressing CRH, CRH-R1 and CRH-R2 were identified after 1 h incubation at room temperature by using the polyclonal goat anti-CRH antibody (C-20; sc-1759, Santa Cruz, CA, USA; dilution 1:50) recognizing a peptide mapping at the carboxy-terminus region of human CRH, the polyclonal goat anti-CRH-R1 antibody (V-14; sc-12381, Santa Cruz, CA, USA; dilution 1:50) recognizing a peptide mapping within an internal region of human CRH-R1, and the polyclonal goat anti-CRH-R2 antibody (N-20; sc-1826, Santa Cruz; CA, USA; dilution 1:50) recognizing a peptide mapping at the amino-terminous region of human CRH-R2.

CRH, CRHR-1 and CRH-R2 detections were performed by the LSAB plus System-HRP (DakoCytomation, Carpinteria, CA, USA), 30 min at room temperature, using diaminobenzidine as chromogen (DAB substrate System, DAKO). To reduce non specific binding, the sections were preincubated with 20% normal swine serum for 30 min at room temperature. Human trophoblast tissue was used as positive control, since human placenta has been shown to express CRH as well as both CRH receptor subtypes [START_REF] Wetzka | Expression patterns of CRH, CRH receptors, and CRH binding protein in human gestational tissue at term[END_REF][START_REF] Gao | Differential Regulation of Prostaglandin Production Mediated by CRH Receptor Type 1 and Type 2 in Cultured Human Placental Trophoblasts[END_REF]. Human placenta was obtained from a healthy subject undergoing elective caesarean section at term of gestation before the onset of labor.

ER, PR, and Ki-67 immunostainings were performed by incubating the sections for 1 h at room temperature with anti-ER (clone 1D5, recognizing ER-alfa; dilution 1:50; Ylem, Rome, Italy), anti-PR (clone 1A6, recognizing PR-A; dilution 1:50; Ylem) and anti-ki-67 (clone MIB-1; dilution 1:100; Slides from all cases were simultaneously processed for immunohistochemistry on the TechMate Horizon automated staining system (DAKO). Negative controls were performed by omitting primary antibodies.

Evaluation of immunohistochemical staining

The analysis of all tissue sections was performed by two of the authors (FOR, EM) without any prior knowledge of clinical and biological parameters. In case of disagreement, sections were submitted to a rejoint evaluation by a multiheaded microscope.

CRH, CRHR-1 and CRH-R2 expressions were evaluated by considering the localization (cytoplasmic and/or nuclear), as well as the percentage of cells exhibiting immunoreaction, ER an PR stainings were scored in a minimum of 300 histologically identified neoplastic cells, showing nuclear reaction. As previously reported [START_REF] Sivridis | Endometrial carcinoma: association of steroid hormone receptor expression with low angiogenesis and bcl-2 expression[END_REF], tumors with ER or PR positive nuclear staining in more than 5% of tumor cells were defined as ER or PR positive, respectively. Scoring of Ki-67 was expressed as the percentage of cells with positive nuclear staining. Tumors with high Ki-67 staining in more than 40% (median value) tumor cells were considered as positive.

Statistical analysis

Fisher's exact test or  2 test were used to analyze the distribution of cases considered positive for the biological parameters examined according to several clinico-pathological features. Disease Free Survival (DFS) was calculated from the date of diagnosis to the date of relapse or the date last seen. Recurrence was considered as any documented relapse of the tumor either locally in the pelvis or systemic dissemination. Medians and life tables were computed using the product-limit estimate by the Kaplan and Meier method [START_REF] Kaplan | Non parametric estimation from incomplete observations[END_REF] and the log-rank test was employed to assess the statistical significance [START_REF] Mantel | Evaluation of survival data and two new rank order statistics arising in its consideration[END_REF]). Statistical analysis was carried out using SOLO (BMDP Statistical Software, Los Angeles, CA).

CRH, CRH-R1 and CRH-R2 immunostaining

Normal glandular endometrial tissues surrounding the tumor display a typical paranuclear/apical pattern of expression (APE) for CRH (Figure 1, panel A). At variance with normal tissues, a diffuse cytoplasmic staining was observed within the tumor cells in all cases (Figure 1, panel B), although this diffuse-staining pattern was not associated to the totality of cells in the tissue, since a percentage of tumor cells maintained an APE pattern (Figure 1, Panel C). Therefore, the percentage of cells presenting with an apical pattern over the total was evaluated according to a score including: 0 (no pattern), 1 (APE <10%), 2 (10% < APE < 50%) and 3 (APE > 50%). All cases with a score 2 or 3 (i.e. with APE >10%) were classified as CRH APE-positive; this occurred in 20 cases (39.2%).

Thirty-seven out of 51 specimens could be assessed for CRH-R1 staining analysis. A diffuse cytoplasmic staining with dot-like reinforcement in the basal cell domain was observed for CRH-R1 in 34 tumor tissues (Figure 2, panel B); a similar but less intense pattern was observed in the normal surrounding tissue (Figure 2, panel A).

Interestingly, CRH-R2 staining in normal endometrial glands was found to be localized at the nuclear level (Figure 3, panel A). The same pattern was also observed in 9 cases of tumors (17.6%) (Figure 3

Correlation with clinico-pathological and biological parameters

As shown in Table 2, no significant difference was documented in the distribution of CRH-APE positivity according to any of the parameters examined.Table 3 shows that a higher percentage of CRH-R1 positive cases was observed in the progesterone receptor (PR)-positive compared to PRnegative subgroup (96.8% versus 60%; p<0.05); a similar trend was observed in the ER-positive versus ER-negative subgroup (96.6% versus 71.4%, respectively), although such difference did not reach the statistical significance. As far as CRH-R2 expression is concerned (Table 2), a higher percentage of CRH-R2 cytoplasmic positive cases was observed in more advanced stage disease compared to FIGO stage I tumors (81.8% versus 44.8%; p value < 0.01). No statistically significant association was found between nuclear CRH-R2 positivity and the parameters examined.

Survival analysis

As of April 2008, the median follow-up was 51 months (range= 8-82). During the follow-up period, recurrence of disease were observed in 5/29 (17.2%) early stage cases, and in 12/51 (23.5%) cases in the whole population. There were no significant differences in DFS according to CRH-APE, CRH-R1, cytoplasmic or nuclear CRH-R2 status (data not shown).

Discussion

To our knowledge, this is the first study evaluating the expression and intra-cellular localization of CRH and its receptors in endometrial cancers; the peculiar pattern of expression observed here A c c e p t e d M a n u s c r i p t strongly suggests a role for CRH receptors and their endogenous ligands in the biology of this malignancy. Moreover, the expression and intra-cellular localization of the two CRH receptor subtypes, CRH-R1 and -R2 has been found associated with specific tumor features such as PR positive status and advanced stage of disease, respectively.

Beyond hypothalamus, CRH has been found to be expressed in other sites of the nervous system, in the gastrointestinal tract [START_REF] Suda | Distribution and characterization of immunoreactive corticotropin-releasing factor in human tissues[END_REF], vasculature [START_REF] Simoncini | Human umbilical vein endothelial cells: a new source and potential target for corticotrophin releasing factor[END_REF], skin (Slominski et al., 1998), as well as in normal human ovaries [START_REF] Mastorakos | Presence of immunoreactive corticotropin-releasing hormone in normal and polycystic human ovaries[END_REF] and endometrium [START_REF] Makrigiannakis | The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium[END_REF][START_REF] Mastorakos | Presence of immunoreactive corticotropin-releasing hormone in human endometrium[END_REF][START_REF] Di Blasio | Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells[END_REF][START_REF] Florio | Paracrine regulation of endometrial function: interaction between progesterone and corticotropin-releasing factor (CRF) and activin A[END_REF]. Moreover, during pregnancy the placenta becomes the major source of CRH, which seems to play a role in the timing of birth [START_REF] Smith | The timing of birth[END_REF]. CRH expression was also found in different human epithelial neoplasia such as thyroid [START_REF] Scopa | Presence of immunoreactive corticotropin releasing hormone in thyroid lesions[END_REF], lung [START_REF] Moody | Corticotropin-releasing factor stimulates cyclic AMP, arachidonic acid release, and growth of lung cancer cells[END_REF], skin [START_REF] Slominski | Characterization of corticotropin-releasing hormone (CRH) in human skin[END_REF], and breast [START_REF] Ciocca | Corticotropin-releasing hormone, luteinizing hormone-releasing hormone, growth hormone-releasing hormone, and somatostatin-like immunoreactivities in biopsies from breast cancer patients[END_REF] cancers. As far as endometrial cancer is concerned, evidence is limited to the finding of CRH expression in Ishikawa cell line [START_REF] Makrigiannakis | The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium[END_REF]. Despite such huge amount of studies addressed to localize CRH expression in normal and tumor parenchymas, no study has been devoted so far to investigate the patterns of intra-cellular CRH distribution.

In this study, we found that CRH is expressed in endometrial cancer cells according to two peculiar cytoplasmic patterns: diffuse and polarized. The diffuse staining of CRH, found in all tumor cases, can be considered as a morphological alteration that might reflect a deregulated intra-cellular trafficking of the protein associated to the neoplastic transformation. Conversely, the apical pattern of expression (CRH-APE), as observed in normal endometrial glands and, to a variable extent, in a discrete proportion of cancer cells, is likely to reflect a morphological aspect of CRH secretion process associated to normal cell polarity. Therefore, it would be interesting to investigate whether such polarized pattern of localization could be associated with a less aggressive tumor phenotype.

In normal human endometrium, the constitutive expression of both CRH and its R1-type receptor (particularly in stroma cells) suggests an autocrine/paracrine action of CRH on endometrial cells, where the peptide elicits progesterone-like cell differentiation [START_REF] Tang | Cyclic adenosine 3',5'-monophosphate induces prolactin expression in stromal cells isolated from human proliferative endometrium[END_REF][START_REF] Ferrari | Corticotropin releasing factor decidualizes human endometrial stromal cells in vitro. Interaction with progestin[END_REF][START_REF] Makrigiannakis | The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium[END_REF][START_REF] Di Blasio | Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells[END_REF][START_REF] Telgmann | Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells[END_REF][START_REF] Florio | Paracrine regulation of endometrial function: interaction between progesterone and corticotropin-releasing factor (CRF) and activin A[END_REF]. As an extension of the physiological role in normal endometrium, in Ishikawa endometrial tumor cells CRH is secreted and acts locally to inhibit basal and estradiol-stimulated cell growth, as well as to promote differentiation via the activation of R1 receptors and the subsequent increase in adenylyl cyclase activity [START_REF] Graziani | CRH inhibits cell growth of human endometrial adenocarcinoma cells via CRH-receptor 1mediated activation of cAMP-PKA pathway[END_REF][START_REF] Graziani | Corticotropin-releasing hormone receptor-1 in human endometrial cancer[END_REF]. Thus, one would expect that the expression of CRH-R1 receptors in human tumors is associated to a phenotype close to the physiological one.

Similar to previous findings by other groups (Di [START_REF] Di Blasio | Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells[END_REF][START_REF] Karteris | Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium[END_REF], we found that CRH-R1 is expressed in the cytoplasm of normal epithelial endometrial cells. Most importantly, CRH-R1 expression was found in a higher percentage of tumors expressing progesterone receptors, indicating that CRH-R1 expression is associated to a more differentiated and less aggressive phenotype. On the same line of evidence, progestins have been found to stimulate the expression and production of endometrial CRH through a cAMP-dependent mechanism [START_REF] Makrigiannakis | The decidualizing effect of progesterone may involve direct transcriptional activation of corticotrophin-releasing hormone from human endometrial stromal cells[END_REF]; in stromal endometrial cells, CRH may mediate the decidualizing effect of progesterone via the binding and activation of CRH-R1 receptors [START_REF] Makrigiannakis | Participation of maternal and fetal CRH in early phases of human implantation: the role of antalarmin[END_REF].

Apart from CRH-R1, CRH anti-tumor activity was also found to be associated to the activation of CRH-R2 receptor subtype, through a mechanism involving VEGF and reduced tumor vascularization [START_REF] Bale | Corticotropin-releasing factor receptor 2 is a tonic suppressor of vascularization[END_REF]. Thus, it was interesting to investigate the expression and localization of such receptor subtype in our paradigm. Human endometrial cells were shown to express CRH-R2 under physiological conditions [START_REF] Karteris | Expression and signalling characteristics of the corticotrophin-releasing hormone receptors during the implantation phase in the human endometrium[END_REF]; in the present study, we could confirm CRH-R2 expression in normal endometrial tissues surrounding the tumor; moreover, we observed an unprecedented nuclear localization, whose functional meaning remains to be established. Conversely, the expression of CRH-R2 in the tumor cell cytoplasm has been more frequently observed in stage II-IV compared to stage I tumors, thus suggesting that high CRH-R2 might identify poor prognosis tumors.

In conclusion, our immunohistochemical investigation first described the comparison between tumor and the surrounding normal tissues in terms of expression and sub-cellular localization of CRH and its receptors; on this regard, normal endometrial tissue is characterized by a paranuclear/apical pattern of expression of CRH, by the presence of CRH-R1 receptors, and the presence and nuclear localization of CRH-R2 receptors. Moreover, our preliminary observations suggest that CRH-R1 is

  antibodies. ER, PR, and Ki-67 detections were performed by the LSAB-2 System HRP (DakoCytomation).

  , panel C), whereas a diffuse moderate/strong cytoplasmic staining was observed in 31 tumor cases (60.7%) (Figure 3, panel B). Placental tissue, used as positive control, stained for CRH (Fig 1 Panel D), CRH-R1 (Fig 2 Panel C), and CRH-R2 (Fig 3 Panel D) in the trophoblast cell layer.

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. CRH-R1 cytoplasmic immunostaining in normal endometrial tissue surrounding the tumor (A); a diffuse cytoplasmic staining with dot-like reinforcement was observed for CRH-R1 in most of endometrial adenocarcinoma analyzed (B). Placental tissue was used as positive control for CRH-R1 immunostaining (D).

Figure 3 .

 3 Figure 3. CRH-R2 immunostaining in endometrial adenocarcinoma. Specific CRH-R2 nuclear staining in normal endometrial glands (A), diffuse cytoplasmic CRH-R2 staining observed in most of tumor tissues (B), tumor case with CRH-R2 nuclear pattern (C). Placental tissue was used as positive control for CRH-R2 immunostaining (D).

  Figure 1.

Figure

  Figure

  Figure 2.

Figure

  Figure

  Figure 3.

Figure

  Figure

Features

  significant P-values <0.01 calculated by Fisher's exact test for proportion. a FIGO stage I versus stage II-IV.

  Distribution for CRH-R1 positivity according to clinico-pathological parameters.

Features*

  Indicates significant P-values <0.05 calculated by Fisher's exact test for proportion.

Table

Table 1 .

 1 Clinico-pathological and biological features of our sample series.

	Features	N°. of cases (%)
	All cases	51
	Age
	< 60	24 (47.1)
	 60	27 (52.9)
	Stage
	I	29 (56.8)
	II	11 (21.6)
	III-IV	11 (21.6)
	Histotype
	Endometrioid	45 (88.3)
	Serous	3 (5.9)
	Clear cell	2 (3.9)
	Undifferentiated	1 (1.9)
	Grade
	1	12 (23.5)
	2	25 (49)
	3	14 (27.5)
	Lymph node status
	Negative	31 (60.7)
	Positive	9 (17.7)
	n.a.	11 (21.6)
	ER
	Positive	36 (70.6)
	Negative	15 (29.4)
	PR
	Positive	42 (82.3)
	Negative	9 (17.7)
	Ki-67
	Low	30 (58.8)
	High	21 (41.2)

Table A

 A

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

Table 2 .

 2 Distribution for CRH-APE, CRH-R1, cytoplasmic and nuclear CRH-R2 positivity according to clinico-pathological parameters.