Jelena Velebit

Helena H Chowdhury

Marko Kreft

Robert Zorec
email: robert.zorec@mf.uni-lj.si

ROSIGLITAZONE BALANCES INSULIN-INDUCED EXO-AND ENDOCYTOSIS IN SINGLE 3T3-L1 ADIPOCYTES

Keywords: Adipocytes, GLUT4, Rosiglitazone, Exocytosis, Endocytosis, Insulin

Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membranebound compartments with the plasma membrane, thus increasing the plasma membrane area.

However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo-and endocytosis are balanced. Here we examined whether Rosi differentially affects the rates of exo-and endocytosis in 3T3-L1 adipocytes. The immunolabelling of GLUT4 revealed the 3.1-fold increase in PM-resident GLUT4 in Rosi-pretreated, insulin-stimulated cells. By monitoring cumulative exocytosis and endocytosis we found that in Rosi-pretreated cells insulin substantially stimulated the rate of exocytosis and to a similar extent also the rate of endocytosis. We conclude that Rosi-pretreatment balances insulin-stimulated exocytosis and endocytosis, which may prevent insulin-mediated adipocyte cell size increase.

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

Introduction

The rate of glucose uptake in muscle and adipose tissue is limited by the density of the glucose transport proteins on the plasma membrane [START_REF] Bryant | The Measurement of GLUT4 Translocation in 3T3-L1 Adipocytes[END_REF][START_REF] Foster | Mechanism and regulation of GLUT-4 vesicle fusion in muscle and fat cells[END_REF]. Insulin regulates glucose uptake in adipocytes and muscle cells by controlling the subcellular distribution of glucose transporters GLUT4 [START_REF] Holman | Moving the insulin-regulated glucose transporter GLUT4 into and out of storage[END_REF][START_REF] Muretta | Insulin Releases Glut4 from Static Storage Compartments into Cycling Endosomes and Increases the Rate Constant for Glut4 Exocytosis[END_REF]. In the basal conditions, GLUT4 is largely excluded from the plasma membrane and is retained within endosomes, the trans-Golgi network, and tubular-vesicular compartments termed GLUT4 storage vesicles (GSVs) [START_REF] Bryant | The Measurement of GLUT4 Translocation in 3T3-L1 Adipocytes[END_REF][START_REF] Foster | Mechanism and regulation of GLUT-4 vesicle fusion in muscle and fat cells[END_REF]. Insulin triggers the redistribution of GLUT4 from intracellular compartments to the plasma membrane which increases the membrane glucose permeability [START_REF] Muretta | Insulin Releases Glut4 from Static Storage Compartments into Cycling Endosomes and Increases the Rate Constant for Glut4 Exocytosis[END_REF]). This appears to be associated with a plasma membrane area increase [START_REF] Chowdhury | Insulin induces a rapid increase in membrane area in single rat adipocytes[END_REF]Velebit et al., 2008). When insulin is removed, GLUT4 is rapidly cleared from the plasma membrane back into intracellular compartments [START_REF] Muretta | Insulin Releases Glut4 from Static Storage Compartments into Cycling Endosomes and Increases the Rate Constant for Glut4 Exocytosis[END_REF]. Thus the distribution of GLUT4 is controlled by insulin-mediated effects on the rate of exo-and endocytosis [START_REF] Muretta | Insulin Releases Glut4 from Static Storage Compartments into Cycling Endosomes and Increases the Rate Constant for Glut4 Exocytosis[END_REF].

Insulin resistance is characterized by a reduced ability of major insulin target tissues such as skeletal muscle, adipose tissue and liver to respond to the action of insulin [START_REF] Petersen | Pathogenesis of skeletal muscle insulin resistance in type 2 diabetes mellitus[END_REF][START_REF] Rattarasarn | Physiological and pathophysiological regulation of regional adipose tissue in the development of insulin resistance and type 2 diabetes[END_REF]Toledo et al., 2006). Thiazolidinediones (TZD) represent a class of oral hypoglycaemic agents that have been shown to improve insulin action and reverse some of the metabolic processes responsible for the development of insulin resistance and, finally, type 2 diabetes in predisposed subjects [START_REF] Bhatia | Insulin resistance and PPAR insulin sensitizers[END_REF][START_REF] Mudaliar | New oral therapies for type 2 diabetes mellitus: the glitazones or insulin sensitizers[END_REF]. Systemically, TZDs lower hyperglycaemia by increasing peripheral glucose utilization and decreasing hepatic glucose production in human and in experimental models of insulin resistance [START_REF] Bhatia | Insulin resistance and PPAR insulin sensitizers[END_REF][START_REF] Olefsky | Treatment of insulin resistance with peroxisome proliferator-activated receptor gamma agonists[END_REF]. At cellular level the insulin sensitizing effects of TZDs are mediated through A c c e p t e d M a n u s c r i p t 4 the peroxisome proliferator-activated receptor γ (PPARγ) [START_REF] Bhatia | Insulin resistance and PPAR insulin sensitizers[END_REF] which is highly abundant in adipose tissue and to a lesser extent in skeletal muscle and liver [START_REF] Auwerx | PPARgamma, the ultimate thrifty gene[END_REF][START_REF] Olefsky | Treatment of insulin resistance with peroxisome proliferator-activated receptor gamma agonists[END_REF]. A representative of TZD, rosiglitazone (Rosi), is a potent agonist of PPARγ and improves the differentiation of 3T3-L1 cells into adipocytes [START_REF] Rangwala | Peroxisome proliferator-activated receptor gamma in diabetes and metabolism[END_REF]Velebit et al., 2008). Furthermore, adipocyte differentiation leads to the enhanced expression of adipocyte-specific genes, such as GLUT4 and insulin receptor substrate-1 (IRS-1), which are important components of the insulin receptor signaltransduction pathway (Ntambi and Young-Chuel, 2000;[START_REF] Okuno | Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats[END_REF]White and Kahn, 1994;Wu et al., 1998). It was shown that the treatment of 3T3-L1 adipocytes with Rosi during differentiation results in the accumulation of GLUT4 at the plasma membrane surface [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF], which is consistent with the concept that TZDs regulate membrane dynamics (Velebit et al., 2008). However, the mechanism by which Rosi affects the increased density of GLUT4 at the plasma membrane remains unclear. It has been shown that insulin induces an increase in net changes of plasma membrane surface area of single 3T3-L1 cells by increasing the rate of exocytosis, via phosphatidylinositol 3-kinase, as in the case of insulin-induced GLUT4 translocation (Velebit et al., 2008), consistent with insulin-induced surface area measurements in adipocytes [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF][START_REF] Chowdhury | Insulin induces a rapid increase in membrane area in single rat adipocytes[END_REF]. Interestingly, the pretreatment of 3T3-L1 cells with Rosi significantly attenuated the insulin-induced increase in membrane area (Velebit et al., 2008), which could be due to a simultaneous insulininduced increase in exo-and endocytosis of Rosi-pretreated cells. To directly address this, we here employed quantitative confocal microscopy and the styryl fluorescent dye FM1-43, a membrane area marker [START_REF] Cochilla | Monitoring secretory membrane with FM1-43 fluorescence[END_REF] to monitor cumulative exocytosis as well as cumulative endocytosis of single 3T3-L1 adipocytes pretreated with Rosi and stimulated with insulin. We also examined subcellular localization of GLUT4 in 3T3-L1 cells by Rosiglitazone maleate was obtained from GlaxoSmithKline (Worthing, UK). Dimethyl sulfoxide (DMSO) was purchased from Merck Schuchardt (Hohenbrunn, Germany). Anti-GLUT4 rabbit polyclonal antibody was obtained from Abcam (Cambridge, UK) and goat anti-rabbit IgG antibody was obtained from Molecular Probes. Vehicle solution contained 1% acidic acid.

Cell Culture

3T3-L1 fibroblasts were grown in DMEM containing 10 % CS, as described previously [START_REF] Rangwala | Peroxisome proliferator-activated receptor gamma in diabetes and metabolism[END_REF]. Briefly, fibroblasts were seeded onto poly-L-lysinecoated coverslips and allowed to grow to confluence. Conversion into adipocytes was induced on day 0 in DMEM containing 10 % CS, 0.25 mM dexamethasone, 0.5 mM IBMX and 174 nM insulin for 2 days (time zero to day 2), and then medium was replaced with DMEM containing 10 % CS and 174 nM insulin for a further 2 days (days 2-4). On day 6 most of the cells were differentiated and cells were transferred to DMEM containing 10 % CS. During experiments, rosiglitazone was added to a final concentration of 10 M, from a 1 mM stock prepared in DMSO. Insulin was prepared as a 1.74 mM stock solution in acidified M rosiglitazone for 24 h (Rosi + Veh), and then treated with vehicle and (4) cells pretreated with 10 M rosiglitazone for 24 h and then stimulated with 0.2 M insulin for an additional 6 min (Rosi + Ins).

Immunofluorescence

To identify the expression of GLUT4 on the adipocytes surface, the primary rabbit anti-GLUT4 antibodies (Abcam, Cambridge, UK) were used and secondary goat anti-rabbit antibody conjugated to fluorescent dye Alexa Fluor 546 (Molecular Probes). 3T3-L1 cells were washed with PBS, fixed in 4 % paraformaldehyde in PBS for 15 min, and then incubated in 0.1 % Triton X-100 for 10 min at room temperature. Non-specific staining was reduced by incubating cells in blocking buffer containing 3 % BSA and 10 % goat serum in PBS at 37°C for 1 h. The cells were incubated with primary antibody (diluted 1:100) in 3 % BSA in PBS at 37°C for 2 hours and secondary antibody (diluted 1:600) in 3 % BSA in PBS for 45 min at 37°C. Subsequently, they were mounted using the Light Antifade kit (Invitrogen). Z-stacks were acquired for each immuno-labelled cell detected using a Zeiss LSM 510 confocal microscope through a planapochromatic oil-immersion objective (x 63, NA=1.4), excited by the 543 nm He/Ne laser line and filtered with the 560 nm low-pass emission filter. Images were quantitatively analyzed using custom software written in Matlab programming language (Math Works, Natick, MA). Eight to 15 markers were manually set to the cell perimeter, and the software interpolated the curve between the markers. The software eroded pixels from the marked perimeter curve to obtain 1.1 m wide zone around the plasma membrane. This procedure resulted in two separated regions of each confocal image: cell periphery and the FM1-43 is an amphiphilic styryl dye that has been used extensively to monitor individual exocytic and endocytic events, as well as vesicle membrane cycling in a variety of cell types, including neuroendocrine cells [START_REF] Cochilla | Monitoring secretory membrane with FM1-43 fluorescence[END_REF] and adipocytes [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF]. From the extracellular fluid these dye molecules reversibly partition into the outer leaflet of surface membrane, but they can not penetrate to the cytoplasm, owing to their charged head groups [START_REF] Henkel | FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals[END_REF]. During exocytosis the luminal vesicle membrane become exposed to external medium and gets stained with FM1-43, thus contributing to the increase in the FM1-43 fluorescence intensity and to the increase in the plasma membrane area. Simultaneously, fluorescently stained membranes get internalized via endocytosis, hence the entire cell fluorescence intensity increase persists and the steady increase in fluorescence signal provides a cumulative record of exocytosis [START_REF] Neves | The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells[END_REF].

After removal of FM1-43 from the medium, dye molecules dissociate from the membrane to the extracellular medium, causing the plasma membrane to destain [START_REF] Henkel | FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals[END_REF]. However cells retain residual fluorescence because some of dye-labeled membrane is internalized by endocytosis. If a stained endosome undergoes exocytosis in dye-free medium, then dye A c c e p t e d M a n u s c r i p t 9 molecules dissociate from this membrane [START_REF] Henkel | FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals[END_REF]. Thus changes in FM1-43 fluorescence intensity are an index of changes in plasma membrane area (Betz, 1996).

Because GLUT4 is stored in the membrane of GLUT4 storage vesicles (GSV; Zorzano, 1989) and insulin induces the GSV translocation to-and fusion with the plasma membrane of adipocytes [START_REF] Smith | Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4[END_REF], this event is inseparably bound to the increase of the plasma membrane area.

To study exocytosis, cells were exposed to FM1-43 for 3-5 min before the addition of insulin. After addition of FM1-43 to the recording medium the final concentration of DMSO was less than 0.5 %. During experiments we added insulin (final concentration 0.2 M) or vehicle, respectively. Images were scanned for 10 min with a 20 s scan interval and analysed quantitatively using the LSM 510 (Time series software, Carl Zeiss). The effect of insulin and vehicle addition onto cells was quantified by measuring changes in FM1-43 fluorescence intensity of entire cell image relative to the resting levels (fluorescence intensity at the time of insulin addition) every 60 s after stimulation.

To establish whether insulin stimulates endocytosis in control and Rosi-pretreated cells, we exposed cells to the following protocol (see Fig.

Immuno-labelling reveals that rosiglitazone-pretreatment of 3T3-L1 cells increases the insulin-induced GLUT4 translocation to the plasma membrane

To determine whether subcellular distribution of GLUT4 is modulated by rosiglitazone (Rosi), we performed GLUT4 immuno-labelling in single 3T3-L1 cells (Fig. 1A), as addressed previously [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF]. Cells were pretreated with 10 M Rosi for 24 h and then stimulated with 0.2 M Ins (Fig. 1A, Rosi + Ins) or with vehicle only (Fig. 1A, Rosi) for 6 minutes. Insulin stimulated cells (Fig. 1A, +Ins) were used to test whether these cells exhibit GLUT4 translocation from the cytoplasm to the plasma membrane in comparison to non-stimulated conditions (Fig. 1A, +Veh). Fluorescence labelling with anti-GLUT4 antibody (see Materials and Methods) revealed the presence of GLUT4 in the cytoplasm and in the plasma membrane of 3T3-L1 adipocytes (Fig. 1A), as previously reported [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF]. To measure the extent of translocation of GLUT4 between the cytoplasm and the plasma membrane, we determined the ratio of fluorescence intensity at the plasma membrane region vs. that of the cytoplasm (Fig. 1B, see Materials and Methods). The ratio of fluorescence intensity in Ins-stimulated cells was 2.1 ± 0.1 (n=12), significantly higher than in control cells to which only the vehicle was applied 1.0 ± 0.1 (n=16, P<0.001; Fig. 1B). Interestingly, the pretreatment of cells with Rosi also resulted in a higher ratio of fluorescence intensity compared to control cells (2.0 ± 0.2, n=9; p<0.001), however not significantly different than in Ins-treated cells (Fig 1B ), consistent with data on cell populations [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF]. The ratio of fluorescence A c c e p t e d M a n u s c r i p t 12 intensity of the (Rosi+Ins)-(pre)treated cells was 3.1 ± 0.2 (n=8), significantly higher than in control cells (P<0.001) and also higher than in only Rosi-pretreated (P<0.01) or only Insstimulated cells (P<0.01). These results on single cells show that Rosi, Ins and Rosi+Ins (pre)treatments increase the fraction of GLUT4 at the plasma membrane vs. the cytoplasm and that the effects of Rosi and insulin are additive.

Rosiglitazone Augments Insulin-Induced Increase in FM1-43 Fluorescence Reporting Cumulative Exocytosis

To study the rate of exocytosis in single cells, we measured the changes in FM1-43 fluorescence intensity of single cell relative to the value at the time of Ins or Veh addition (Fig 2A). Once 3T3-L1 cells were placed into the recording medium, we started to scan cells with the argon laser to determine the resting fluorescence. To establish whether Ins addition stimulates membrane dynamics, we added a bolus into the chamber (see Materials and methods) to reach a final concentration of 0.2 M Ins with non-pretreated and Rosipretreated cells, respectively. In controls we applied the Veh only. Fluorescent images on Figure 2A show live 3T3-L1 adipocytes, before (left images) and after (right images) vehicle (+Veh) or insulin (+Ins) additions to non-pretreated cells and to Rosi-pretreated cells (Rosi+Veh and Rosi+Ins). In the presence of Ins, the fluorescence intensity at the cell perimeter increased significantly vs. controls (Fig. 2A, right), consistent with previous experiments on primary adipocytes [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF][START_REF] Chowdhury | Insulin induces a rapid increase in membrane area in single rat adipocytes[END_REF]. Some of the dye is internalised by endocytosis, therefore we measured the fluorescence intensity of entire cell to obtain a report of cumulative exocytosis [START_REF] Neves | The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells[END_REF]. In controls, the addition of Veh as a bolus resulted in a small change in the slope of the FM1-43 fluorescence intensity signal (+Veh). However, the application of Ins to nonpretreated cells augmented the rate of FM1-43 fluorescence intensity increase (+Ins), which is consistent with an increase in membrane area, as reported [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF]Velebit et al., 2008). Importantly, the rate of FM1-43 fluorescence intensity signal increased significantly in Rosi-pretreated cells vs. non-pretreated cells (Rosi+Veh), almost following the signal increase in Ins-stimulated cells. The application of Ins to Rosi-pretreated cells promoted further the increase in FM1-43 fluorescence intensity (Rosi+Ins) and was significantly higher than in only Ins-stimulated cells. significantly different from controls (P<0.001). Six minutes following the addition of Ins into the chamber with Rosi-pretreated cells, fluorescence intensity increased by 72.4 ± 7.0 % and the rate of fluorescence intensity increase, corresponding to a slope coefficient of the regression line, was 11.2 ± 0.6 %/min (Fig. 2B ii, full squares, n=6), significantly higher than in controls (P<0.001), in Rosi-pretreated cells (P<0.001) and in cells stimulated with Ins only (P<0.001). These results are consistent with the view that the Rosi-pretreatment augments the Ins-induced increase in cumulative exocytosis in 3T3-L1 cells.

Rosiglitazone pretreatment increases Cumulative Endocytosis

In another set of experiments, we determined the rate of cumulative endocytosis in 3T3-L1 cells in basal and in stimulated conditions. After the wash of FM1-43 from the chamber medium, 3T3-L1 cells retained a residual fluorescence, indicating FM1-43 dye internalisation by endocytosis. Between the two FM1-43 exposures insulin or vehicle were added to the cells (Fig. 3A) and the % change between both residual fluorescence was taken as the read out of the rate of cumulative endocytosis (in %/min). We compared the residual fluorescence intensity (a.u.) of cells following the two sequential exposures/removals of FM1-43 (Fig. 3B). After second incubation with FM1-43 dye the mean intensity of residual fluorescence significantly increased compared to first incubation in each group of cells (Fig. 3B), demonstrating a cumulative endocytosis. The results are summarized in Fig. 3C. In controls (+Veh) the rate of cumulative endocytosis was 2.1 ± 0.3 %/min (n = 7), whereas in Rosi-pretreated cells (Rosi+Veh) it was 4.5 ± 0.7 %/min (n = 7), significantly higher than in controls (P<0.01). The application of insulin (+Ins) resulted in a significant increase in the rate of endocytosis compared to controls (P<0.01) and was 7.5 ± 1.8 %/min (n = 4), however A c c e p t e d M a n u s c r i p t 15 not significantly different than in Rosi-pretreated cells (Rosi+Veh). Importantly, in cells pretreated with 10 M Rosi for 24 h, subsequent insulin application (Rosi+Ins) promoted the rate of endocytosis to 13.3 ± 1.6 %/min (n = 4), which was significantly higher than in control cells, in Rosi-pretreated cells and in cells stimulated with Ins (P<0.01).

Discussion

In this study we examined the effects of insulin and rosiglitazone on the regulation of membrane area dynamics in single 3T3-L1 adipocytes. Specifically, we examined (1) the effect of Rosi-pretreatment on GLUT4 translocation to the plasma membrane by quantitative confocal microscopy, (2) the effect of Ins application on cumulative exocytosis and endocytosis and (3) the effect of acute (24 h) rosiglitazone pretreatment on Ins-mediated plasma membrane dynamics.

Rosiglitazone increases the plasma membrane GLUT4 density via altered membrane dynamics

Translocation of GLUT4 from intracellular pool to the plasma membrane involves insulin-induced exocytosis of GLUT4 bearing vesicles [START_REF] Ezaki | Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents[END_REF][START_REF] Oka | Photo affinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin[END_REF]Whitesell and Abumrad, 1985). Increased exocytosis is associated with an increase in plasma membrane surface area [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF][START_REF] Chowdhury | Insulin induces a rapid increase in membrane area in single rat adipocytes[END_REF]Velebit et al., 2008). Results in this study (Fig. 1) confirm that insulin stimulation increases the density of GLUT4 on the plasma membrane.

Rosi is a potent antidiabetic drug which improves insulin sensitivity and glycaemic status [START_REF] Phillips | Once-and twice-daily dosing with rosiglitazone improves glycemic control in patients with type 2 diabetes[END_REF]. In addition, it was shown that pretreatment with Rosi and other TZDs leads to the reduction of the adipocyte cell size [START_REF] Johnson | Prolonged decrease of adipocyte size after rosiglitazone treatment in high-and low-fat-fed rats[END_REF]Velebit et al., 2008). This may be due to alterations in membrane dynamics; however, the mechanism of cell size reduction was not studied yet. Our results obtained at cellular level show that in 3T3-L1 adipocytes the fraction of membrane GLUT4 is increased following the pretreatment with Rosi (Fig. 1A, Rosi+Veh), which is consistent with recently reported observations [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF]. Moreover, the Rosi-pretreatment enhanced the Ins-induced fraction of GLUT4 A c c e p t e d M a n u s c r i p t 17 at the plasma membrane (Fig. 1B, Rosi+Ins), as was also shown in cell populations [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF]. While it was suggested that the increased fraction of GLUT4 at the plasma membrane may be due to altered membrane dynamics (Velebit et al., 2008), and/or endosomal recycling [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF], this was not studied directly. The results on Figures 2 and3 suggest that the increased localization of GLUT4 at the plasma membrane may be due to Rosi-mediated discrete effects on the rate of exo-and endocytosis, since Ins significantly increased the fluorescence intensity of the FM1-43 (Fig. 2A) and application of Rosi (24 h) amplified the effect of Ins.

A slow steady increase in FM1-43 fluorescence observed in resting cells (Fig. 2A) is likely due to constitutive exocytosis [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF]. In support of membrane internalization after a continuous slow process of exocytosis, we observed that cells exposed to FM1-43 remained fluorescent even after removing FM1-43 from the medium (Fig. 3A).

This indicates that some of the FM1-43 dye molecules are captured within cells by endocytosis.

Rates of exocytosis and endocytosis after rosiglitazone and insulin treatment

The effect of insulin

Here we show that insulin application increases the rate of exocytosis by a factor of 4.0 vs. controls (Table 1) in 3T3-L1 adipocytes, similar to the increase elicited by insulin in primary adipocytes [START_REF] Chowdhury | Rapid insulin-induced exocytosis in white rat adipocytes[END_REF]. This increase in rate of exocytosis is probably associated with GLUT4 density regulation [START_REF] Czech | Fat targets for insulin signaling[END_REF] and/or secretion of e.g. adipsin, Acrp30 [START_REF] Ahima | Adipose tissue as an endocrine organ[END_REF][START_REF] Kitagawa | Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes[END_REF][START_REF] Scherer | A novel serum protein similar to C1q, produced exclusively in adipocytes[END_REF] andother adipokines. An increased density of plasma membrane transporters by insulin (Figure 1), could also be due to an inhibition of endocytosis, but our results show that endocytosis is not inhibited by insulin. In contrast, insulin increased the rate of cumulative endocytosis by a A c c e p t e d M a n u s c r i p t 18 factor of 3.6 vs. controls (Table 1). The insulin-mediated increased rate of endocytosis may be associated with the internalization of insulin and the insulin receptor [START_REF] Di Guglielmo | Insulin receptor internalization and signaling[END_REF]. However, since GLUT4 continuously cycles between vesicles and the plasma membrane [START_REF] Saltiel | Insulin signalling and the regulation of glucose and lipid metabolism[END_REF], it is conceivable that insulin increases cell surface density of the transporter not only by increasing the exocytotic rate but also by increasing the size of the recycling GLUT4 pool [START_REF] Muretta | Insulin Releases Glut4 from Static Storage Compartments into Cycling Endosomes and Increases the Rate Constant for Glut4 Exocytosis[END_REF], implicating a potential role for endocytosis in regulating cell surface levels of the transporter in adipocytes. Although the rates of exo-and endocytosis do not appear significantly different (P = 0.73; Table 1), the fold increase compared to controls is greater in exocytosis, which is in accordance with our previously published data, where insulin was shown to stimulate plasma membrane area increase in 3T3-L1 adipocytes (Velebit et al., 2008).

The effect of rosiglitazone

Although it is generally thought that TZDs mediate their insulin-sensitizing effects via PPARγ, PPARγ-independent pathways have also been suggested [START_REF] Blackmore | Actions of the novel antidiabetic agent englitazone in rat hepatocytes[END_REF][START_REF] Furnsinn | Chronic and acute effects of thiazolidinediones BM13.1258 and BM15.2054 on rat skeletal muscle glucose metabolism[END_REF][START_REF] Preininger | Acute troglitazone action in isolated perfused rat liver[END_REF][START_REF] Chawla | PPAR-gamma dependent and independent effects on macrophage-gene expression inlipid metabolism and inflammation[END_REF].

Our results show that cells exposed to rosiglitazone for 24 hours exhibit an augmented rate in exocytosis (Fig. 2B), by a factor of 2.9 vs. controls (Table 1). We measured also an increase in the rate of endocytosis (Fig. 3C) by a factor of 2.2 vs. controls (Table 1). It seems that rosiglitazone augments constitutive exocytosis as well as constitutive endocytosis, thus consequently increases the membrane turnover, indicating altered adipocyte biology due to rosiglitazone action. It is possible that rosiglitazone selectively affects the synthesis of some molecules involved in the translocation of GLUT4, as it was demonstrated that in 3T3-L1 adipocytes a member of TZD family, troglitazone, increases the expression of annexin II, which is involved in vesicular transport [START_REF] Huang | Annexin II is a thiazolidinedione-responsive gene involved in insulin-induced glucose transporter isoform 4 translocation in 3T3-L1 adipocytes[END_REF]. These and other molecules, in rosiglitazone-pretreated cells. Moreover, a recent study demonstrates that rosiglitazone induces an increase in the expression of the insulin receptor [START_REF] Martinez | Rosiglitazone increases cell surface GLUT4 levels in 3T3-L1 adipocytes through an enhancement of endosomal recycling[END_REF], which could imply an activation of the insulin signalling cascade.

Synergistic effect of rosiglitazone and insulin

Rosiglitazone, a PPARγ agonist, intensifies insulin signalling in adipose tissue (Ahima and Flier, 2002), however it attenuates the insulin-stimulated increase in membrane surface area (Velebit et al., 2008). It was suggested that in rosiglitazone-pretreated cells insulin stimulates a rapid increase in exocytosis that is associated with a similar increase in the rate of endocytosis, since both processes appear to be controlled by insulin (Velebit et al., 2008). In the current study, the application of insulin in Rosi-pretreated cells (Rosi+Ins) stimulates a rapid increase in the rate of exocytosis by a factor of 6.1 vs. controls (+Veh) and by a factor of 1.5 vs. Rosi-nontreated cells (+Ins) (Table 1). These results indicate that rosiglitazone invigorates insulin-stimulated exocytosis. Insulin application in Rosi-pretreated cells also increased the residual fluorescence intensity, reporting cumulative endocytosis by a factor of 6.3 vs. controls (+Veh) and by a factor of 1.8 vs. Rosi-nontreated cells (+Ins). These results report that the insulin-stimulated endocytosis, as shown previously [START_REF] Chowdhury | Insulin induces a rapid increase in membrane area in single rat adipocytes[END_REF], is highly amplified with rosiglitazone pretreatment. While the absolute rates of exo-and endocytosis are comparable (Table 1; P=0.54), the comparison of rates of exo-and endocytosis (Table 1) shows that Rosi-pretreatmet affected more strongly the insulin induced-increased rate of endocytosis (1.8 fold) than the rate of exocytosis (1.5 fold).

If the overall rates of exo-and endocytosis are linearly related to the dynamics of exoand endocytosis of vesicles bearing GLUT4 solely, then one would expect that rosiglitazone would not enhance the membrane resident fraction of GLUT4. In contrast, the data in Figure The results of this study provide evidences that rosiglitazone may balance the insulininduced increase in the rate of exocytosis and endocytosis, which may prevent the insulininduced cell size increase.

Pathophysiological Implications

An insulin-dependent imbalance between the rates of exo-and endocytosis may promote the increase in cell size during lipogenesis. Consistent with this it was reported that vagally mediated insulin hypersecretion is associated with an increased adipocyte size and with insulin resistance [START_REF] Del Prato | Effect of sustained physiologic hyperinsulinaemia and hyperglycaemia on insulin secretion and insulin sensitivity in man[END_REF][START_REF] Jeanrenaud | Hyperinsulinemia in obesity syndromes: its metabolic consequences and possible etiology[END_REF]. While a net insulindependent increase of plasma membrane area recorded [START_REF] Chowdhury | Insulin induces a rapid increase in membrane area in single rat adipocytes[END_REF]Velebit et al., 2008) is consistent with an increase in cell size mediated by insulin, it is presently not clear if and how an imbalance between exo-and endocytosis may induce insulin resistance.

Activation of PPAR in white adipose tissue increases the number of small adipocytes, and reduces the number of large adipocytes [START_REF] Rangwala | Peroxisome proliferator-activated receptor gamma in diabetes and metabolism[END_REF]. Small adipocytes are more sensitive to insulin [START_REF] Lowell | PPARgamma: an essential regulator of adipogenesis and modulator of fat cell function[END_REF] and an increased number of small adipocytes in white adipose tissue can alleviate insulin resistance [START_REF] Lowell | PPARgamma: an essential regulator of adipogenesis and modulator of fat cell function[END_REF]. Present results suggest that rosiglitazone pretreatment not only increases the membrane turnover in adipocytes, but also balances out the insulin-mediated difference between the rates of exocytosis and endocytosis. This mechanism may prevent increase in cell size of adipocytes observed in

  physiological significance of our results is discussed in the context of adipocyte cell size regulation. modified Eagle's medium (DMEM), Calf serum (CS), dexamethasone, isobutylmethylxanthine (IBMX), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), KCl, D-glucose, NaH 2 PO 4 , NaHCO 3 , Na 2 ATP, phosphate buffered saline (PBS), bovine serum albumin (BSA), goat serum, paraformaldehyde, triton X-100 and insulin were purchased from Sigma (St. Louis, MO). CaCl 2 × 6H 2 O was obtained from Riedel De Haen (Seelze-Hannover, Germany). MgCl 2 was purchased from Kemika (Zagreb, Croatia).

  (pH 2, using glacial acetic acid, approx. 1 %). Differentiated 3T3-L1 cells were divided into four experimental groups for each type of experiments: (1) control cells, vehicle addition (+Veh); (2) cells stimulated with 0.2 M insulin for 6 min (+Ins); (3) cells pretreated with 10

  The mean fluorescence intensity of these regions was calculated and then the ratio of fluorescence intensity between plasma membrane area and cytoplasm (I m /I c ) was determined.2.4. Time-laps confocal microscopy and fluorimetryDifferentiated 3T3-L1 adipocytes were placed in a recording chamber on the confocal microscope (LSM 510, Zeiss, Oberkochen, Germany). The recording medium consisted of (in mM): NaCl 131.8, CaCl 2 1.8, KCl 5, MgCl 2 2, HEPES/NaOH 10, D-glucose 10, NaH 2 PO 4 0.5, NaHCO 3 5; pH 7.2. The recording medium also contained 4 M FM1-43 (Molecular Probes, Eugene, Ore., USA), diluted previously in DMSO to obtain a 1 mM stock.

  4A): cells were exposed to FM1-43 fluorescent dye for 1 minute to establish a baseline for individual cell. After that FM1-43 dye was washed out from the recording chamber and replaced with extracellular solution for 5 minutes. Cells were then stimulated with insulin and incubated again with FM1-43 for 6 minutes and again washed out. After removal of FM1-43, cells retained a residual fluorescence, because FM1-43 dye was internalized by endocytosis. After both FM1-43 washouts z-stacked images of cells were acquired. The effect of insulin and vehicle addition onto cells was quantified by measuring changes in FM1-43 residual fluorescence intensity of the cell interior after second incubation relative to the residual fluorescence of cell interior after first incubation with FM1-43.

  A c c e p t e d M a n u s c r i p t 10 The fluorescence images of FM1-43 stained cells were acquired through a planapochromatic oil-immersion objective (x 40, NA = 1.3), excited by the 488 nm argon laser line and filtered with the 505 nm low-pass emission filter. All experiments were performed at room temperature. Data are given as mean ± SEM.

  Figure 1.

Figure 2 .

 2 Figure 2.

  The results are summarized in Figure2B. Mean changes in FM1-43 fluorescence intensity of entire cell intersection were calculated for every minute interval after application of the drug. With linear regression analysis of data points (dotted lines) we calculated the rate of FM1-43 fluorescence intensity increase. Regression lines are drawn according to the equation: y= ax + n; where y is mean fluorescence intensity [%], a is the slope coefficient [%/min], x is time [min] and n is resting fluorescence intensity [%]. All slope coefficients are significantly different from zero (p<0.001) and report change in the fluorescence intensity per unit of time, which reflects the rate of cumulative exocytosis. In controls, following the application of Veh, FM1-43 fluorescence increased by 11.7 ± 1.6 % in 6 min, with a slow steady rate of 1.8 ± 0.1 %/min (Fig 2B i, open circles, n=6), as evidenced by the regression analysis. In Rosi-pretreated cells the fluorescence intensity increased by 31.4 ± 5.9 % (6 min after the vehicle addition) and corresponding regression line revealed a steady increase with a slope coefficient of 5.3 ± 0.1 %/min (Fig 2B ii, open squares, n=6), significantly higher from A c c e p t e d M a n u s c r i p t 14 controls (P<0.001). Ins-application resulted in a fluorescence signal increase of 45.8 ± 6.0 % with the rate in fluorescence signal of 7.3 ± 0.5 %/min (Fig. 2B i, full circles, n=7),

Figure 3 .

 3 Figure 3.

  in exocytosis, may be involved in the increased translocation of GLUT4

  the dynamics of GLUT 4 vesicles, the respective processes of exo-and endocytosis appear to depart from this prediction. Therefore, one can conclude that while the overall dynamics of exo-and endocytosis of adipocytes is balanced by rosiglitazone pretreatment, the traffic of GLUT4-bearing vesicles appears to augment the membrane associated fraction of GLUT4. This could be achieved by many mechanisms, including the selective enhancement of exocytosis, a reduction of endocytosis or the combination of both.

  Thus this may explain in part the role of rosiglitazone in alleviating insulin resistance symptoms. .G, Sniderman, A.D., Kelley, D.E., 2006. Influence of hepatic steatosis (fatty liver) on severity and composition of dyslipidemia in type 2 diabetes. Diab. Care 29, 1845-1850. Velebit, J., Kovačič, P.B., Prebil, M., Chowdhury, H.H., Grilc, S., Kreft, M., Jensen, J., Isenović, E.R., Zorec, R., 2008. Rosiglitazone modulates insulin-induced plasma membrane area changes in single 3T3-L1 adipocytes. J. Membr. Biol. 223, 141-149. White, M.F., Kahn, C.R., 1994. The insulin signaling system. J. Biol. Chem. 269, 1-4. Whitesell, R.R., Abumrad, N.A., 1985. Increased affinity predominates in insulin stimulation of glucose transport in the adipocyte. J. Biol. Chem. 260, 2894-2899. Wu, Z., Xie, Y., Morrison, R.F., Bucher, N.L., Farmer, S.R., 1998. PPARγ induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPγ during the conversion of 3T3 fibroblasts into adipocytes. J. Clin. Invest. 101, 22-32. Zorzano A., Wilkinson W., Kotliar N., Thoidis G., Wadzinkski B.E., Ruoho A.E., Pilch P.F., 1989. Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J. Biol. Chem. 264:12358-63.

Figure 1 .

 1 Figure Legends:

Figure 2 .

 2 Figure 2. Time-dependent changes of FM1-43 fluorescence intensity reflecting cumulative exocytosis. (A) Single cross-section confocal micrographs of FM1-43 -stained adipocytes before (left panels) and at the end of recording (right panels) in control (vehicle

  Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Acknowledgements

This work was supported by the Grant #P3 310 of Slovenian Research Agency, the COST (European Cooperation in Science and Technology) action BM0602, and support from Ad Futura (Slovene Human Resources Development and Scholarship Fund). We thank for the generous sample of rosiglitazone to GlaxoSmithKline.


Tareq Mukattash

Ahmed F Hawwa

PhD Karen Trew

PhD, CPsychol James C Mcelnay
email: j.mcelnay@qub.ac.uk

Healthcare professional experiences and attitudes on unlicensed/off-label paediatric prescribing and paediatric clinical trials

Keywords: Unlicensed medicines, off-label medicines, clinical trials, children

Objectives: To investigate the knowledge and views of a range of healthcare professionals (consultant paediatricians, GPs, community pharmacists and paediatric nurses) regarding the use of unlicensed/off-label medicines in children and the participation of children in clinical trials.

Methods: A regional study in which a survey instrument with 39 items was issued to 500 randomly selected GPs, all community pharmacists (n=512), 50 hospital consultants and 150 paediatric nurses in Northern Ireland.

Results: Approximately half (46.5%) of the 1,212 healthcare professionals approached responded to the questionnaire. The majority of respondents indicated their familiarity with the term unlicensed (82.9%) or off-label (58.6%) prescribing with the most frequently quoted reason for such prescribing being younger age (33.6%). Apart from community pharmacists, most respondents reported having gained their knowledge through personal experience. Even though a large percentage of respondents expressed concerns about the safety (77.8%) or efficacy (87.9%) of unlicensed/off-label prescribing in children, only 30.7% reported informing parents/guardians about such use of medicines in children. In addition, only 56% of respondents believed that unlicensed/off-label medicines should undergo clinical trials in children. Overall, 28.4% of respondents (20.1% of GPs, 41.4% of community pharmacists, 27.7% of paediatric nurses and 94% of consultant paediatricians) indicated their willingness to be actively involved in, and recruit their patients for, paediatric clinical research.

Introduction:

Even though, it is well documented that children are not small adults, [START_REF] Wildt | The effect of age on drug metabolism[END_REF][START_REF] Sammons | Recommendations of the ad hoc group for the development of implementing guidelines for Directive 2001/20/EC relating to good clinical practice in the conduct of clinical trials on medicinal products for human use[END_REF] many of the medicines currently used to treat children are based upon extrapolation of adult data. [START_REF] Budetti | Ensuring safe and effective medications for children[END_REF][START_REF] Steinbrook | Testing medications in children[END_REF] This is due to the fact that many medicines are only tested in and licensed for adult patients. [START_REF] Collier | Paediatric prescribing: using unlicensed drugs and medicines outside their licensed indications[END_REF] A review of 30 studies carried out between 1984 and 2004, showed that 11-37% of drugs prescribed to children in the community setting, 16-62% prescribed in general paediatric wards and up to 80% in neonatal intensive care units are either unlicensed or used in an off-label manner (i.e. prescribed outside their product license with respect to age, dose, route of administration or indication). [START_REF] Pandolfini | A literature review on off-label drug use in children[END_REF] Despite this wide-spread use of unlicensed/off-label medicines in children, only 40% of General Practitioners (GPs) admit to unlicensed/off-label prescribing. [START_REF] Ekins-Daukes | Off label prescribing to children: attitudes and experience of general practitioners[END_REF] In addition, a study has shown that in a cohort of 95 French paediatricians, 92% of the paediatricians did not recognise the off-label status of the medicines they prescribed. [START_REF] Chalumeau | Off label and unlicensed drug use among French office based paediatricians[END_REF] Another study conducted recently in 4 Italian paediatric hospitals, found a discordance between the existing prescribing and physicians' perceptions of off-label prescribing. This was explained by the fact that paediatricians did not consider some of the most commonly prescribed drugs in their everyday practice as being off-label. [START_REF] Marchetti | The awareness among paediatricians of off-label prescribing in children: a survey of Italian hospitals[END_REF] Off-label/unlicensed prescribing for children is a global problem, however, recent major research initiatives in the USA and Europe are beginning to address the issue of the need for a greater evidence base for the safe and effective use of medicines in this age group. [START_REF] Hoppu | Paediatric clinical pharmacology: at the beginning of a new era[END_REF] In the meantime, off-label/unlicensed prescribing often represents the most rational use of medicines if no approved alternatives for children with comparable benefit-risk ratio are available. [START_REF] Hoppu | Paediatric clinical pharmacology: at the beginning of a new era[END_REF] Very often some data are available on paediatric use, which ranges from scarce to adequate for market authorisation, but even when adequate data are available, these have not been submitted by a pharmaceutical company to the regulatory authorities for evaluation and no age appropriate formulations are available. [START_REF] Hoppu | Paediatric clinical pharmacology: at the beginning of a new era[END_REF] There is growing concern about the safety and efficacy of unlicensed/off-label medicine use in children particularly due to the lack of long-term safety and efficacy data in this vulnerable group and due to evidence suggesting an increased risk of adverse drug reactions (ADRs) and treatment failure. [START_REF] Impicciatore | Incidence of adverse drug reactions in paediatric in/out-patients: a systematic review and meta-analysis of prospective studies[END_REF][START_REF] Choonara | Unlicensed and off label drug use in children: implications for safety[END_REF][START_REF] Turner | Adverse drug reactions to unlicensed and off label drugs on paediatric wards: a prospective study[END_REF] The ethical obligation of providing the best available treatment to children coupled with a lack of availability of licensed paediatric products, highlights the need for an increased number of clinical trials in children. Performing such trials, however, requires special attention to the various challenges faced in this population. [START_REF] Sammons | Ethical issues of clinical trials in children: a European perspective[END_REF] To date, limited data have been published on the views and experiences of different healthcare professionals on these matters. The aim of the present study was, therefore, to investigate the knowledge and views of a wide range of healthcare professionals (including consultant paediatricians, GPs, community pharmacists and paediatric nurses) regarding the use of unlicensed/off-label medicines in children and the participation of children in clinical trials.

Methods:

A survey instrument comprising 39 questions (Appendix 1) was designed after extensive literature review. The questionnaire focused primarily on the use of unlicensed and off-label medicines in children and consisted of five sections addressing different topics of interest together with a separate section concerning participant details and demographics (to allow examination of variability in responses according to participant characteristics): The first section focused on the experiences and views of healthcare professionals regarding the use of unlicensed and off-label medicines in children while the second section focused on parental involvement in decision making regarding the medicine being prescribed for their child. The third section addressed the information sources that healthcare professionals use when prescribing, dispensing and administering unlicensed/off-label medicines to children.

The fourth section focused on paediatric clinical trials while the final section focused on dose-related issues when prescribing unlicensed and off-label medicines in children.

Information (definitions and examples

) regarding the unlicensed/off-label use of medicines in children was provided as an integral part of the questionnaire to guide respondents who were not familiar with the terminology.

Prior to the full study, the questionnaire was piloted in a small number of healthcare professionals (n=20) (Pharmacists and Doctors); these results were not included in the final analysis. The respondents indicated that the questionnaire was clear and easy to understand. The questionnaire was then posted to GPs, community pharmacists and hospital based paediatric nurses and e-mailed to consultant paediatricians across Northern Ireland. The questionnaires were mailed to 500 randomly selected GPs (http://allen.netcom.co.uk/Sampling.htm) and all community pharmacists (n=512). The recipients were allowed four weeks to return the completed questionnaire. A letter was also sent to paediatric nursing units of all hospitals in Northern Ireland. The letters were sent to the head of each ward asking them to reply if they were willing to have their nursing staff take part in this study and if this was the case, they were also asked to indicate the number of questionnaires that would be required in each unit. Questionnaires (n=150) were sent to these units which agreed to take part in the study giving them a four week period to complete the questionnaire.

Finally, questionnaires were e-mailed to all consultant paediatricians in Northern Ireland (n=50) giving them four weeks to complete the questionnaire. This survey was not sent to hospital pharmacists due to the small number of specialist paediatric pharmacists in Northern Ireland. Following data collection, responses were coded and entered into a customised database in SPSS, version 15, for statistical analyses (Chi-square test and sign tests). Consultation with the Office of Research Ethics Committees in Northern Ireland (ORECNI) confirmed that the study did not require formal review by the National Health Service (NHS) ethics committee. The study, however, received ethical approval from the School of Pharmacy, Queen's University Belfast Research Ethics Committee.

Results:

Demographics

Out of the 1,212 distributed questionnaires, 563 (46.5%) were returned completed and 32 uncompleted. A total of 59.8% of GPs, 32.8% of community pharmacists, 72.0% of consultant paediatricians and 43.3% of paediatric nurses replied. The majority of respondents were female (59.2%) and aged between 20-40 years old (60.8%).

Knowledge and views about unlicensed/off-label medicines

The majority of respondents (467; 82.9%) indicated that they were familiar with the term, unlicensed medicines while 330 (58.6%) indicated they were familiar with the term off-label medicines prior to receiving the questionnaire. There was a significant difference in the familiarity with unlicensed/off-label prescribing among the different sectors of health care professionals (P<0.05) with community pharmacists being most familiar with the term unlicensed medicines (93%) and consultant paediatricians most familiar with the term, offlabel medicines (83.3%). When asked how they became familiar with the terminology of unlicensed and off-label medicines, 51.7% of respondents said they had gained their knowledge through their professional experience rather than through undergraduate (24%) or postgraduate (16.5%) training. Separate analysis for community pharmacists, however, indicated that they had gained their knowledge mainly through their undergraduate studies (50.0%) rather than through their experience (39.0%) or postgraduate studies (33.1%), the latter data being significantly different (P<0.05) from corresponding results for other groups.

Approximately one quarter (24.5%) and one third (31%) of the respondents reported actually prescribing, dispensing or administering unlicensed and off-label medicines, respectively, to children in their practice during the four weeks that preceded answering the questionnaire. They reported that the reasons for off-label prescribing were younger age (33.6%), lower than recommended dose (30.7%), higher than recommended dose (29.1%), different indication (29.1%) or different route of administration (24.2%) from that of the licensed product. No significant differences in the perceived reasons for off-label prescribing were found among the different healthcare professionals (P>0.05). In addition, the majority of respondents reported infants (1 to 23 months of age) to be most likely to receive unlicensed (40.9%) or off-label (35.0%) medicines. On the other hand, children from 2 to 11 years old and children from 12 to 18 years old were, according to the respondents, less likely to receive unlicensed (22.1%) or off-label (33.0%) medicines (Table 1).

When asked to rank the most important ways by which unlicensed and off-label medicine use in children could be reduced, the majority of respondents (60%) ranked making more appropriate formulations available for younger children as a 'very important' approach, followed by increasing clinical trials for new drugs (38.5%) and finally increasing clinical trials for existing drugs (28.6%). There were no significant differences between different groups of health care professionals with regards to this subject (P>0.05).

Views about the safety and efficacy of unlicensed/off-label medicines

The majority of respondents had major and/or minor concerns regarding the safety (77.8%) and the efficacy (87.9%) of unlicensed and off-label medicine use in children. Safety and efficacy concerns were more frequently raised by pharmacists and GPs when compared with consultant paediatricians and paediatric nurses (89.0%, 93% vs 83.3%, 64.4% for safety and 81.7%, 80.3% vs 69.4%, 61.5% for efficacy; P<0.05; Chi Square test). Concerns about the safety of unlicensed and off-label medicines in children were addressed again in a further question which examined whether respondents felt that the use of unlicensed and off-label medicines increased the likelihood of ADRs when compared with licensed medicines in children. Results for each group of respondents are presented in Figure 1. Discussing their own practice, approximately one quarter (23.8%) of respondents admitted to having experienced treatment failure while 11.2% to having observed ADRs when prescribing, dispensing or administering unlicensed/off-label medicines in children. Interestingly, there was a significant relationship between those participants who reported experiencing treatment failures (23.8%) and those who reported lower than recommended doses as the main reason for off-label use (30.7%; P<0.01). In addition, there was a significant correlation between those who reported experiencing cases of ADRs (11.2%) and those who reported higher than recommended dosing as being the main reason for off-label use (29.1%; P<0.05). Treatment failures and adverse effects were reported by more consultant paediatricians when compared to the other respondent groups (i.e. 41.7%, 28.9%, 15.9%, 10.7% for treatment failure vs 25.0%, 11.7%, 8.5%, 7.6 for ADRs; same categories as Figure 1). When asked about which is more problematic with regard to safety, approximately one quarter of respondents (26.8%) thought that the use of unlicensed medicines was more problematic when compared to off-label medicines. However, almost half the respondents (48.5%) felt that the use of unlicensed and off-label medicines is equally problematic with regard to safety.

Dosing-related issues and information sources

The vast majority (93.4%) of respondents said they double checked doses of medicines used in children if a calculation was required in deciding on the dose. Interestingly, 78.3% of respondents had come across a dosing error in a paediatric prescription in their professional practice. Almost half of respondents (43.7%) felt that licensing more medicines for use in children would decrease dosing errors in children. However, GPs and community pharmacists had stronger views than consultants and paediatric nurses in this regard (47.0% and 55.5% vs 8.3% and 18.5%, respectively). When asked about the various information sources they used when dealing with medicines in children, respondents reported that the British National Formulary for Children (BNFc) was the most commonly used, followed by the British National Formulary (BNF), Table 2. In addition, they stated that, of all sources, the BNFc contained the most appropriate information to support their practice.

Communication with parents and guardians

Although 85.4% of respondents agreed that parents/guardians should be told when an unlicensed or off-label medicine was prescribed for their children, only 30.7% of respondents reported informing parents and guardians when such medicines were used.

Only 1.1% of respondents said that they request written consent from parents or guardians, while 45.1% said that they request verbal consent when informing parents about the unlicensed/off-label use of medicines. GPs were the group that most frequently claimed informing parents (41.9%) and asking for verbal consent (39.3%).

Overall, respondents felt that parents should be informed by the doctor (95.7%), pharmacist (2.3%) or nurse (0.5%) within the hospital setting while in primary care, the providers of the information were selected as follows: the doctor (94.0%) and the community pharmacist (4.6%).

Clinical trials in children

When asked about the need for more paediatric clinical trials to address the issue of unlicensed/off-label medicine use, 56% of the respondents believed that medicines used in an unlicensed/off-label fashion should undergo clinical trials in children. This percentage was reasonably consistent for all healthcare professional groups (i.e. 55.6%, 52.7%, 64.9%, 49.3%; same categories as Figure 1). Moreover, approximately half of respondents (54.7%) agreed that pharmaceutical companies should be forced to carry out research on new medicines in children. However, only 28.4% of respondents indicated that they would be willing to be actively involved in, and recruit their own patients for, paediatric clinical research. Consultant paediatricians (94.4%) reported most willingness to help with clinical trials when compared with other health care professional groups (Figure 2).

Respondents were also asked if they would consent for their own children to participate in clinical trials. No significant difference was found between those who had children of their own and those who did not (P>0.05; Chi Square test). However, this willingness was influenced by the assumed health status of the child and whether the medicine was new or an existing medicine used in unlicensed/off-label manner (Figure 3).

Discussion:

The lack of alternate approved medicines for children, and the lack of paediatric formulations even if data on paediatric pharmacology of a drug are available, mean that offlabel and unlicensed prescribing continues to be a necessary feature of contemporary paediatric practice. To our knowledge, this is the first study to contemporaneously assess the levels of knowledge and views on unlicensed/off-label medicines in children in a range of healthcare professionals (consultant paediatricians, GPs, community pharmacists and paediatric nurses); 1,212 healthcare professionals throughout Northern Ireland were approached in this survey, with approximately half of them responding.

The majority of consultants, GPs and community pharmacists were familiar with the concept of unlicensed/off-label prescribing. The most commonly reported reason for such prescribing was 'use at a younger age than recommended'. This finding was consistent with earlier studies examining views/attitudes of GPs and community pharmacists in the UK [START_REF] Ekins-Daukes | Off label prescribing to children: attitudes and experience of general practitioners[END_REF][START_REF] Stewart | Attitudes and experiences of community pharmacists towards paediatric off-label prescribing: a prospective survey[END_REF] and matches actual data collected in a survey of UK hospital-based prescribing. [START_REF] Turner | Unlicensed and off label drug use in paediatric wards: prospective study[END_REF] In the community setting in the UK, [START_REF] Ekins-Daukes | Paediatric off label prescribing in general practice[END_REF] however, 'lower dose than recommended' has been shown to be the most frequent cause for off-label prescribing while younger age than recommended is amongst the least frequent reason for this mode of medicine use.

Apart from the pharmacists, most respondents (including GPs) reported having gained their knowledge through personal experience rather than via their undergraduate or postgraduate training, a similar situation to that reported in an earlier study. [START_REF] Ekins-Daukes | Off label prescribing to children: attitudes and experience of general practitioners[END_REF] This reliance on personal experience highlights a significant deficiency in current healthcare undergraduate and postgraduate education.

It was interesting to note that a very low percentage of healthcare professionals inform parents or seek their consent when an unlicensed or off-label medicine is prescribed, dispensed or administered to a child. This finding coincides with a recent study performed in Germany that showed a limited and surprisingly low knowledge of off-label prescribing among parents of chronically ill children. [START_REF] Lenk | Off-label, off-limits? Parental awareness and attitudes towards off-label use in paediatrics[END_REF] In an earlier public opinion survey carried out in Northern Ireland, a significant percentage of parents/guardians reported that they would refuse the use of such medicines or ask for the prescription of a licensed alternative if they were informed about the unlicensed use of medicines in their child. In addition, the majority of participants in that latter study (92.1%) felt that parents should be told if their child was being prescribed a medicine that had not been fully studied or licensed in children. [START_REF] Mukattash | Public awareness and views on unlicensed use of medicines in children[END_REF] A large number of respondents expressed concerns regarding the safety and efficacy of unlicensed/off-label medicines and reported cases of treatment failure and side effects of such agents during their practice. Paediatric nurses were the least concerned about safety/efficacy issues when compared with the other groups of healthcare professionals. This could be attributed to them being very accustomed to treating large numbers of children, many of whom will receive medicines outside the licensed recommendations.

Another interesting result was the fact that the reported experiences of treatment failure in this study were higher than experiences of ADRs following the use of unlicensed/off-label medicines in children. This may reflect clinicians being over cautious in using appropriately high doses. This was supported by the fact that experiences of treatment failures were associated with 'lower than recommended' dosing being identified as the main reason for off-label use.

The vast majority of respondents (93.4%) claimed that they double checked the doses of medicines used in children when a dosage calculation is required. In doing this, more than three quarters of respondents reported that they had come across a dosing error in a paediatric prescription in their professional practice. Concern about this high incidence of error detection is compounded by the fact that the literature shows that medication errors in children receiving unlicensed/off-label medicines have three times the potential to cause harm when compared with similar errors in adults. [START_REF] Fortescue | Prioritizing strategies for preventing medication errors and adverse drug events in pediatric inpatients[END_REF] Surprisingly, only half of the respondents believed that unlicensed/off-label medicines should undergo clinical trials in children despite their concerns about their safety and efficacy. These findings were consistent with a recent survey of the attitudes of hospital based paediatricians [START_REF] Mclay | A prospective questionnaire assessment of attitudes and experiences of off label prescribing among hospital based paediatricians[END_REF] in Scotland but in contrast with those expressed by Scottish GPs who were more convinced of the need for appropriate clinical trials in children. [START_REF] Ekins-Daukes | Off label prescribing to children: attitudes and experience of general practitioners[END_REF] In order to conduct such clinical trials there needs to be an increased willingness of both clinicians and parents to become involved in this type of research. Such willingness was found in the present study to be very much higher in consultant paediatricians when compared with the other professional groups (Figure 2). It has been shown that the willingness of parents to consent for their children to take part in clinical trials is associated with the worsening of the child's health status and when children can directly benefit from the clinical research. [START_REF] Lenk | Off-label, off-limits? Parental awareness and attitudes towards off-label use in paediatrics[END_REF][START_REF] Mukattash | Public awareness and views on unlicensed use of medicines in children[END_REF] The present study showed that this also holds true for healthcare professionals who are themselves parents.

Overall the research indicated that still much remains to be achieved in the area of both managing off-label and unlicensed medicine use and in creating the environment in which research in children to improve the evidence base is integrated into routine practice.

APPENDIX 1. THE USE OF UNLICENSED AND OFF-LABEL MEDICINES IN CHILDREN

Definition: The International Conference on Harmonisation (ICH) has defined children to be individuals of both sexes who range between 0 and 18 years of age. This age range has been used in this questionnaire.

For each question please circle the option which you agree with, unless the question tells you to choose more than one option.

I. Experiences and views regarding medicines for children

Figure 1 :Figure 2 :

 12 Figure 1: Respondents' concerns regarding the likelihood of unlicensed and off-label medicines increasing the level of adverse drug reactions in children (P<0.05; Chi Square test)

Figure 3 :

 3 Figure 3: Respondents (parents vs. non-parents) responses regarding enrolling their child in a clinical trial based on their child's health status and whether the medicine to be tested was a new medicine or an existing medicine used in an unlicensed or off-label manner.

2 . 3 . 4 .II. Parent and guardian involvement 17 . 18 . 19 .

 234171819 Before completing this questionnaire, were you familiar with the definition for unlicensed medicines? Before completing on this questionnaire, were you familiar with the definition for off-label medicines? When did you first hear about the use of unlicensed or off-label medicines? (a) in my undergraduate studies (b) in my post graduate studies (c) through my experience in my work (d) the terminology was new to me 5. During the last 4 weeks, how many times did you prescribe/dispense/administer an unlicensed medicine to a child in your practice. __________ times (please insert number; if none insert 0) In your view, should parents or guardians should be routinely told when an unlicensed or offlabel medicine is prescribed/dispensed/ given to their child. Do you inform parents or guardians when an unlicensed or off-label medicine for their child has been prescribed/ dispensed/ administered? (a) All of the time (b) Some of the time (c) Never (d) Do not come across unlicensed and off-label medicines for children in my practice. Do you request informed, written consent from parents or guardians before prescribing/ dispensing/ administering unlicensed or off-label medicines to their child? (a) Yes (b) No (c) Do not come across unlicensed and off-label medicines for children in my practice 20. Do you request informed, verbal consent from parents or guardians before prescribing/ dispensing/ administering unlicensed or off-label medicines to their child? (a) Yes (b) No (c) Do not come across unlicensed and off-label medicines for children in my practice 21. Who do you think has the main responsibility to inform parents or guardians that their child is being treated using an unlicensed or off-label medicine in the hospital setting? (a) The doctor (b) The nurse (c) The pharmacist (d) Parent or guardian does not need to be informed 22. Who do you think has the main responsibility to inform parents or guardians that their child is being treated using an unlicensed or off-label medicine in primary care? (a) The doctor (b) The pharmacist (c) Parent or guardian does not need to be informed 23. I think that it would unnecessarily worry parents or guardians if I told them that a medicine that their child was being treated with was being used in an unlicensed or off-label manner. prescribing/dispensing/administering medicines for children, to what extent do you use the following information sources: (Please also indicate whether you feel each source contains sufficient information for you needs)

Table 1 : Unlicensed and off-label medicine prescribing, dispensing and administration to children by the different health care professionals ** Consultant paediatrician General practitioner Community pharmacist Paediatric nurse

 1

		U.L.*	O.L.*	U.L.*	O.L.*	U.L.*	O.L.*	U.L.*	O.L.*
	Neonates, including
	preterm and full
	term babies from	52.8%	41.6%	36.9%	33.2%	36.6%	30.5%	23.1% 24.6%
	birth to 28 days
	Infants from one to
	23 months	36.1%	34.2%	38.6%	33.5%	47.6%	37.8%	44.6% 33.9%
	Children from two to
	11 years	11.1%	19.4%	17.8%	24.2%	13.4%	34.7%	24.6% 36.9%
	Children from 12 to
	18 years	0.0%	2.8%	6.7%	9.1%	2.4%	0.0%	7.7%	4.6%
	* U.L.: unlicensed medicine, O.L.: off-label medicine
	**The data refer to activities in the last four weeks
	There was no statistically significant difference between the rates of use between the different professionals group
	(P> 0.05)

Table 2 : Information sources used by healthcare professionals when prescribing, dispensing or administering unlicensed and off-label medicines to children Very frequently Frequently Rarely Never

 2

	Contains sufficient
	information

1. In your opinion, how safe are medicines that are prescribed for use in children

  Prior to drugs being marketed they undergo trials to ensure safety and efficacy in adult volunteers and patients. If the results are positive, and meet strict criteria, a license is granted to market the medicine. Treatment of children may involve use of medicines without a license (unlicensed).

				?
	(a) Extremely safe	(b) Safe	(c) Unsafe	(d) Extremely unsafe (e) I don't know

Unlicensed medicines include those which: are prepared from basic ingredients within the pharmacy; are modifications of a licensed medicinal product, for example, an oral liquid preparation made from crushed tablets.

Medicines can also be prescribed for children off-label. Off-label means that the medicine is licensed, however, it is being used outside the license recommendations with respect to the dose, indication, route of administration or recommended age, for example, increased number of puffs of an inhaler in an attempt to improve efficacy; use of intramuscular vitamin K injection by the oral route. Such use of medications is, however, necessary in paediatric practice to offer the best treatment for sick children.

Conclusion:

The use of unlicensed and off-label medicines remains a major issue in paediatric medicine. Until such times as more licensed medicines are available for children, clear guidance should be developed to allow consistency in practice across the healthcare professionals who are involved with such medicines in their routine practice.

Conflict of Interests:

The authors declare that they have no conflict of interest. Lower than recommended dose used e.g. Extended dosage interval to take account of possible decreased elimination in children. Higher than recommended dose used e.g. the use of a higher dose in an attempt to improve effectiveness.

Use at a younger age than recommended e.g. the use of medicines in infants when the medicines are only licensed for use in older children.

Use of medicine via a different route of administration

e.g. Oral administration of intramuscular preparations due to non-availability of an oral liquid formulation.

Use of medicine for a different indication (from the licensed indication)

e.g. the use of anti-inflammatory medicines to treat pain in children.

In general, do you have concerns about the efficacy of unlicensed and off-label medicine use in children?

(a) Yes, a lot more concerns than for licensed medicines (b) Yes, some more concerns than for licensed medicines (c) No, no more concerns than for licensed medicines

In general, do you have concerns about the safety of unlicensed and off-label medicine use in children?

(a) Yes, a lot more concerns than for licensed medicines (b) Yes, some more concerns than for licensed medicines (c) No, no more concerns than for licensed medicines

V. Dosing Issues

Medicine use in children may require extrapolating the dose based on the weight or surface area of the child, from the dose used in adults.

Do you routinely double check doses of medicines used in children if a calculation was required in deciding on the dose to be given?