Journal of Neuroendocrinology 20, 79–84 ORIGINAL ARTICLE ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd

Chronic Central Administration of Apelin-13 Over 10 Days Increases Food Intake, Body Weight, Locomotor Activity and Body Temperature in C57BL/6 Mice

A. Valle,* à N. Hoggard, A. C. Adams,à P. Roca* and J. R. Speakmanà *Grup de Metabolisme Energe`tic i Nutricio´, Departament de Biologia Fonamental i Cie`ncies de la Salut, Institut Universitari d’Investigacio´ en Cie`ncies de la Salut (IUNICS), Universitat de les Illes Balears, Palma de Mallorca, Spain. Division of Obesity and Metabolic Health, Rowett Research Institute, Aberdeen Centre for Energy Regulation and Obesity (ACERO), Aberdeen, UK. àACERO, School of Biological Sciences, University of Aberdeen, Aberdeen, UK.

Journal of The peptide apelin has been located in a wide range of tissues, including the gastrointestinal tract, stomach and adipose tissue. Apelin and its receptor has also been detected in the arcuate Neuroendocrinology and paraventricular nuclei of the hypothalamus, which are involved in the control of feeding behaviour and energy expenditure. This distribution suggests apelin may play a role in energy homeostasis, but previous attempts to discern the effects of apelin by acute injection into the brain have yielded conflicting results. We examined the effect of a chronic 10-day intracerebro- ventricular (i.c.v.) infusion of apelin-13 into the third ventricle on food intake, body temperature and locomotor activity in C57BL ⁄ 6 mice. Apelin-13 (1 lg ⁄ day) increased food intake significantly on days 3–7 of infusion; thereafter, food intake of treated and control individuals converged. This convergence was potentially because of progressive conversion of apelin-13 to [Pyr1]apelin- 13 which has a four-fold lower receptor binding affinity at the orphan G protein-coupled recep- tor, APJ. Locomotor activity was also higher in the apelin-treated mice, especially during the nocturnal peak, when most feeding occurs, and the first hours of the light phase. Body temper- ature was also elevated during this increased period of activity, but was otherwise unaffected. Apelin-13-infused animals gained more weight than the saline-infused controls, suggesting the elevated locomotor activity did not offset the increased food intake. Elevated locomotion and the consequent increases in body temperature were probably secondary effects to the increased food intake. These results suggest that apelin-13 may play a central role in the control of feed- ing behaviour and is one of only two peripheral ligands known to stimulate rather than inhibit

Correspondence to: intake. As apelin production is elevated during obesity, this may provide an important feed-for- Professor John R. Speakman, ward mechanism exacerbating the problem. Antagonists of the apelin receptor may therefore be Aberdeen Centre for Energy useful pharmaceuticals in the treatment of obesity. Regulation and Obesity (ACERO), School of Biological Sciences, Key words: apelin, intracerebroventricular infusion, food intake, weight gain, activity, body tem- University of Aberdeen, Aberdeen perature, telemetry. AB24 2TZ, UK (e-mail: [email protected]). doi: 10.1111/j.1365-2826.2007.01617.x

Apelin is a peptide identified as the endogenous ligand of the lin-17, measured as the extracellular acidification rate in cultured orphan G protein-coupled receptor, APJ (1). This peptide is derived cells expressing the APJ receptor (1). Although little is know about from a 77-amino-acid precursor, which is processed to several the physiological role of apelin, the highly conserved sequence of molecular forms in different tissues, including apelin-36, apelin-17 apelin and APJ suggest an important physiological role (4). To date, and apelin-13 (2, 3). Apelin-13 is the main form circulating in several studies have shown that apelin is involved in the regulation plasma, and it has greater biological activity than apelin-36 or ape- of cardiovascular function (5–8) and fluid homeostasis (9, 10) 80 A. Valle et al. because systemic administration of apelin lowers blood pressure Measurement of motor activity and body temperature and increases water intake (6). Spontaneous motor activity and body temperature were recorded continu- Apelin and its receptor are widely expressed, particularly in the ously using an implanted passive transmitter (E-Mitter, Mini-Mitter, Bend, OR, central nervous system, heart, lung, testis, ovary, kidney, mammary USA). For implantation of the transmitter in the abdominal cavity, animals gland, gastric mucosa and adipose tissue (3). In the gastric mucosa, (n ¼ 12) were anaesthetised with isoflurane and an approximately 1-cm inci- apelin is involved in gastric cell proliferation, exocrine and endo- sion through the skin and abdominal muscle layer was made. After implanta- crine functions (11, 12). Recently, it was demonstrated that apelin tion, the peritoneal muscle and skin layers were closed with absorbable is secreted by adipose tissue, increased in obesity and regulated by sutures (5-0 Silk, Ethicon, Gargrave, England, UK). Each animal was returned to its home cage with ad libitum food and water for the duration of the both insulin and tumour necrosis factor-a (13–15). In the rat hypo- study. The transmitter obtains power from a radiofrequency field produced by thalamus, apelin mRNA expression is detected in several areas, an energiser ⁄ receiver placed below the cage of the animals. Locomotor activ- including the paraventricular, arcuate and supraoptic nuclei, which ity and body temperature were measured 1 min using a Windows-based data are involved in the control of feeding behaviour and circadian acquision system (VitalView, Mini-mitter, Bend, OR, USA). rhythms (16, 17). The reported distribution of apelin and the APJ receptor in the hypothalamus, gastric mucosa and adipose tissue has led to the hypothesis that apelin may have a role in the control Chronic i.c.v. apelin-13 infusion of feeding behaviour and energy homeostasis. Several previous After a 10-day recovery period from transmitter implantation, animals were studies have addressed this hypothesis; however, the results anaesthetised with isoflurane and placed in a stereotactic frame. The surgi- obtained are contradictory. Acute intracerebroventricular (i.c.v.) cal area was shaved and cleaned with povidone-iodine and alcohol swab. A administration of apelin-13 decreased food intake in both fed and dorsal incision of approximately 1.5 cm running laterally on the scalp was made, the skull was exposed and cleaned and then drilled at coordinates: starved rats (18). The same effects were observed when apelin was anterioposterior, )0.82 mm; lateral, 0.30 mm from the bregma. The can- administered acutely i.c.v. nocturnally, but apelin-12 stimulated nula was then implanted at a depth of ) 2.5 mm below the dura. The can- intake following acute i.c.v. administration during the day-time also nula was secured in position by dental cement. Afterwards, an Alzet osmotic in rats (19). A further study of rats reported a slight increase in minipump (Alzet model 2002; Durect Corporation, Cupertino, CA, USA) filled food intake 2–4 h after acute i.c.v. administration, although this with saline or apelin-13 (H-4566; Bachem, Merseyside, UK) was implanted was not significant on the accumulated 24-h food intake (9). As far by insertion under the skin between the scapulae, and was connected by as energy expenditure is concerned, one report in the rat has catheter to the i.c.v. cannula through a subcutaneous tunnel. The pumps were loaded to deliver 1 lg ⁄ 7 ll ⁄ day of apelin-13 (n ¼ 6 animals) or shown that apelin increased core body temperature and locomotor 7 ll ⁄ day of saline alone (n ¼ 6 animals). The dose of apelin-13 we used activity (20); however, the extent to which these are linked and was based on previous acute i.c.v. studies (9, 18–20). These previous studies dependent on feeding behaviour changes remains unclear. Previous have used single injections of 1–30 nmol of apelin each day into rats confusion over the effects of apelin on feeding behaviour may weighing 250–300 g. For an average dose of 10 nmol, which previously reflect variation in the doses and time points of apelin administra- generated observable effects on food intake and thermogenesis, this is tion and potential handling artefacts during acute i.c.v. administra- equivalent to 0.05 lg apelin-13 ⁄ g body weight ⁄ day. Because our mice tion. Taking into account the confusion that currently exists in the weighed approximately 20 g, we also dosed at 0.05 lg apelin-13 ⁄ g body weight ⁄ day. The main difference, however, was that this dose was delivered literature, we determined the effects of chronic i.c.v. administration in our animals by slow infusion over 24 h whereas, in the previous studies, of apelin-13 on feeding behaviour, body weight, activity and body it was delivered as a single bolus injection. All animals were allowed to temperature using implanted transmitters, allowing us to noninva- recover and telemetry recordings were resumed to monitor activity and body sively monitor these traits using the mouse as a standard model. temperature continuously for a further 10 days, along with daily measures of food intake and body temperature. All observations were made blind of the treatment. At the end of the experiment, animals were killed and loca- Materials and methods tion of the cannula tip in the third ventricle was confirmed histologically.

Animals Telemetry data sampling and analysis Animals were kept in accordance with UK Home Office guidelines. Female C57BL ⁄ 6 mice were purchased from Harlan UK Ltd (Bicester, Oxon, UK) and Data were sampled from the transmitters every 1 min throughout the individually housed under controlled conditions of temperature (22 C) and experiment. Data were pooled and averaged (temperature) or accumulated under a 12 : 12 h light ⁄ dark cycle (lights on 07.00 h). At the start of experi- (activity) every 15 min for analysis and 30 min for presentation. Resting ments, they were approximately 12 weeks old and weighed 20–23 g. Rodent temperature refers to temperature values obtained when the animal was chow (RM3 pellets, Special diet services, BP Nutrition, Norwich, UK) and inactive at least for 15 min. water were provided ad libitum. The exact composition of this diet has been presented previously (21). It contains approximately 50% carbohydrate, 20% protein and 12% fat, by energy, and the balance is made up of indigestible Apelin stability ash and fibre. Absorption efficiency is approximately 76.5% (22). Body We performed an in vitro experiment to evaluate the stability of apelin-13 weights of the mice were measured daily throughout the experiment. Food at body temperatures. Apelin-13 was dissolved in water and incubated at intake was measured from the amount of food missing from the food hop- 36.9 1 C in a sealed glass vessel for 12 days. Each day, two 5-ll aliqu- per each day. Losses into the sawdust have been previously quantified as ots of the solution were removed (at 09.30 h and 16.30 h) and analysed by less than 2% each day (23).

ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 20, 79–84 Effects of chronic central apelin-13 infusion 81 mass spectrometry. Discussion with the company suggested that in solution increase in food intake, by about 15% with respect to the baseline 1 apelin-13 is unstable and forms [Pyr ]apelin-13 which involves autoreaction values. The increase in apelin-stimulated food intake was present of the N-terminal glutamine to form pyroglutamine and a loss of molecular from days 3–7, after which a slight downward trend appeared. Fol- weight by 16 due to removal of the NH group. The expected molecular 2 lowing surgery, both groups of mice increased in body weight over weight was 1550 for apelin-13 and 1534 for [Pyr1]apelin-13. Both these compounds have biological activity at the APJ receptor but the receptor the duration of the infusions. However, the apelin-treated group binding affinity for apelin-13 is approximately four-fold higher than had significantly (P ¼ 0.045) greater weight gain, especially on [Pyr1]apelin-13. Matrix-assisted laser desorption ionisation was carried out days 6–8 (Fig. 2). After this period, the body weights were not sig- using the Applied Biosystems Voyager DE PRO (Applied Biosystems, Foster nificantly different, consistent with the decline in the difference in City, CA, USA) running in reflectron mode. To prepare the sample, each food intake. This suggested that the efficacy of the infused apelin 0.5 ll sample was spotted onto the Teflon coated target plate and before it was diminished after about 7–8 days. dried 0.5 ll of CHCA (5 mg ⁄ ml 70% acetonitrile, 30% water and 0.1% tri- fluoroacetic acid) was added to the spot. This was allowed to air dry before placing in the instrument. The spectra were obtained as a result of 200 Effect of chronic i.c.v. apelin-13 on feeding behaviour- shots of the laser. Spectra were collected from 900–2000 Mr to allow for the presence of other degradation products of the peptide. The effects of i.c.v. apelin-13 administered to C57BL ⁄ 6 mice on feeding behaviour was monitored during the 12 h of maximal food intake ⁄ activity using digital video cameras. Over this 12-h period, Statistical analysis apelin-13 had no significant effect on the duration of each meal Results are presented as means SEM. Data were analysed by two-way (Fig. 3A), the frequency of meals (Fig. 3B) or the total time spent ANOVA with repeated measures, followed by post-hoc least significant differ- feeding (Fig. 3C). ence tests. P < 0.05 was considered statistically significant.

Effect of i.c.v. apelin-13 on mean body temperature and Results activity during light-dark cycle

Effect of i.c.v. apelin-13 over 10 days on food intake Apelin-treated animals showed significantly (ANOVA: P < 0.05) higher values of body temperature and activity during the nocturnal peak To determine whether chronic administration of apelin had an of activity ⁄ temperature as well as during the first hours of the light effect on food intake, we administered apelin to C57BL 6 mice over ⁄ phase (Fig. 4). Motor activity contributes to thermogenesis. To a 10-day period and measured daily food intake (Fig. 1). Surgical remove the effect of activity on body temperature, we calculated intervention (day 0) produced a decrease in food intake that recov- the body temperature including only values when animals were ered to presurgical levels on day 2 (Fig. 1). On day 3, both groups inactive for at least 15 min. As illustrated by Fig. 4(A,C), the increase showed an increase in food intake, probably in compensation for in body temperature was completely dependent on the increase in the post surgical decrease. On day 4, control animals returned to motor activity because there was no effect of treatment on body baseline levels whereas apelin-treated animals showed a significant temperature once the effects of activity had been removed (Fig. 4C).

ANOVA: A, D 6 ANOVA: A, D * * 5 * * 3 * * * * 4 2 3 intake (g)

Food 2 Body weight gain (g) Saline 1 Saline 1 Apelin Apelin

0 0 –4 –3 –2 –10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Time (days) Time (days)

Fig. 1. Effect of chronic i.c.v. apelin (1 lg ⁄ day) or saline administered to Fig. 2. Effect of chronic i.c.v. apelin (1 lg ⁄ day) or saline administered to C57BL ⁄ 6 mice on daily food intake (g ⁄ day). Food intake was measured daily C57BL ⁄ 6 over 10 days on change in body weight relative to baseline. Data from day )4 prior to i.c.v. (day 0) to day 10 (n ¼ 6 per group). Data are are presented as mean SEM (n ¼ 6 per group). Two-way ANOVA with presented as mean SEM. Two-way ANOVA with repeated measures revealed repeated measures revealed a significant effect of treatment (A, P ¼ 0.04) a significant effect (P < 0.01) of treatment (A) and day (D). The asterisk indi- and day (D, P < 0.01). The asterisk indicates time points where differences cates time points where differences are significant (P < 0.05) are significant (P < 0.05)

ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 20, 79–84 82 A. Valle et al.

(A) (B)(C) 10 15 1.8 NS NS NS 8 12 1.5 Saline

1.2 Apelin 6 9 0.9 Hours

Minutes 4 6 0.6 Number of meals 2 3 0.3

0 0 0 Duration of meal Number of meals Total time

Fig. 3. Effect of chronic i.c.v. apelin (1 lg ⁄ day) or saline administered to C57BL ⁄ 6 mice over 3 days (days 5–7) on meal duration (A), meal frequency (B) and total meal duration (C). Animals were recorded with a video camera over 12 h (from 16.00 h to 04.00 h, lights off 19.00 h). Data are presented as mean - SEM from four animals per group. No significant changes were found (NS) (P > 0.05).

(A) Body temperature (B) Activity (C) Resting temperature 38.5 40 38.5 ANOVA: A, T ANOVA: A, T ANOVA: T 38 38 30 37.5 37.5

37 20 37

36.5 36.5 Activity (AU) Temperature (ºC) Temperature (ºC) 10 36 Saline 36 Apelin Saline Saline Apelin Apelin 35.5 0 35.5 01:00 03:00 05:00 07:00 09:00 17:00 19:00 21:00 01:00 03:00 05:00 07:00 09:00 17:00 19:00 21:00 23:00 11:00 13:00 15:00 15:00 11:00 13:00 23:00 01:00 03:00 07:00 09:00 05:00 17:00 19:00 21:00 15:00 11:00 13:00 23:00 Time (h) Time (h) Time (h)

Fig. 4. Effect of chronic i.c.v. apelin (1 lg ⁄ day) or saline on mean body temperature (A), locomotor activity (B) and resting temperature (C) administered to C57BL ⁄ 6 mice during the light and dark phases. Horizontal bar indicates the dark phase. Data are presented as mean SEM averaged over a 4-day periods (from day 6–9) (n ¼ 6 per group). Two-way ANOVA with repeated measures was used: A, Significant effect of treatment; T, significant effect of time (P < 0.05).

Because this elevated activity corresponds to the time when these Discussion animals predominantly feed, we suggest that the primary effect of apelin is to stimulate food intake. This requires an elevation in Apelin and its receptor are expressed in several hypothalamic nuclei physical activity, which generates a corresponding effect on body critical for feeding behaviour and energy homeostasis, such as the temperature. supraoptic, arcuate and paraventricular nuclei (17, 24). Furthermore, apelin has been shown to be expressed in, and secreted into the circulation from, peripheral tissues, such as the gastric mucosa (12) Apelin stability and adipose tissue (13). These tissues have been extensively demon- Mass spectra (Fig. 5A) revealed a peak at Mr 1550 corresponding to strated to modulate appetite by feedback signals such as leptin (19) the expectation for apelin-13 (Mr 1550). A second peak at 1534 or ghrelin (25, 26). Moreover, apelin is increased in hyperinsulin- probably reflected the primary breakdown product in solution emia-associated obesity disorders in which feeding behaviour and [Pyr1]apelin-13. We quantified the size of this peak relative to the energy balance are altered (13, 14). These findings have led to the main 1550 peak on each day throughout the 12-day experiment. suggestion that apelin could have a role modulating feeding behav- There was a progressive increase in the size of the 1534 peak rela- iour and energy homeostasis. tive to the 1550 peak over time (Fig. 5B), suggesting that the ape- In the present study, we have demonstrated that apelin-13, lin-13 was being continuously converted into [Pyr1]apelin-13, as when administered chronically by i.c.v. infusion, increased food anticipated. However, there was no indication of other breakdown intake in C57BL ⁄ 6 mice. By contrast, acute i.c.v. injection of apelin- products in the spectra. 13 decreased food intake in fed and starved rats (18) and similar

ª 2008 The Authors. Journal Compilation ª 2008 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 20, 79–84 Effects of chronic central apelin-13 infusion 83

(A) effects immediately following acute injection may be confused by 100 1550.8038 3.0E+4 handling artefacts. Alternatively, chronic administration may be more prone to cause desensitisation. 90 1552.7900 During chronic infusion, apelin-13 produced a sustained and highly significant increase of food intake accompanied by higher 80 body temperature and locomotor activity during the nocturnal feed- 70 1553.7791 ing period. These latter effects on locomotion and body temperature were in agreement with previous reports (20). This effect on feeding 60 behaviour is consistent with the high expression of APJ receptor in 50 1534.6975 paraventricular and arcuate nuclei, areas where cell populations % Intensity 1533.7708 with appetite inhibiting and stimulating characteristics are localised 40 (27).

30 1554.7490 The effect on body temperature that we recorded was stimulated completely by the increased locomotor activity because there was 20 1536.6616 no elevation of body temperature during periods when the animals 1510.9547 were inactive. Moreover, because the increased activity occurred 10 1555.7138 1512.9698 1549.9043 1678.7689 during the period when the mice have the majority of their food 0 0 intake, it is likely that elevated activity was secondary to the ele- 1399.0 1479.2 1559.4 1639.6 1719.8 1800.0 Mass (m/z) vated food intake (i.e. the mice had to move around to feed), with the increased activity having a further effect on body temperature. (B) % signal intensity of 1534 peak relative to 1550 peak This interpretation is supported by the fact that the energy expen- diture associated with increased activity was insufficient to offset 120 the elevated caloric intake and hence the mice given apelin-13 100 gained more weight than saline-infused controls. If, on the other hand, apelin-13 had stimulated activity and the food intake 80 increase was secondary to that, no effect or even a loss of body weight might be anticipated. The impact on body weight was not 60 sustained beyond day 8 of infusion and, at that time, food intake also started to converge between treatment and control animals. 40 We demonstrated in a separate in vitro experiment that apelin-13 was progressively converted to [Pyr1]apelin-13 over 12 days when 20 maintained in water at body temperature. However, we saw no evi- dence of other breakdown products. The decline in the effects of 0 the infusion on food intake and body weight over time may be 0 100 200 300 400 1 Time (hours) because the APJ receptor binding affinity for [Pyr ]apelin-13 is approximately four-fold lower than apelin-13 (receptor binding data Fig. 5. (A) Mass spectrum for solution containing apelin-13 showing the provided by BaChem, Merseyside, UK). expected main peak around Mr 1550 and a secondary peak around Mr 1534 Most of the peripheral peptides involved in food intake regula- that probably reflects the main breakdown product. (B) Relative size of the tion discovered to date have inhibitory effects on food intake. The breakdown product peak (1534) to the main peak (1550) over time (h) in a sample of apelin-13 dissolved in water and incubated at approximately only exception is ghrelin (28, 29). Our data suggest that apelin-13 36.9 C. The breakdown product progressively increased over time, suggest- is also a peptide that stimulates food intake. The main significance ing that apelin-13 is unstable at body temperatures over periods of several of this discovery is that the production of apelin, similar to other days. adipokines such as leptin, is increased in obesity. As obese humans become insensitive to the appetite suppressing effects of leptin, the continued stimulation of intake by apelin may be an important effects where found when apelin-12 was administered nocturnally feed-forward effect that contributes to the problem. Antagonists to to rats, but contrasting stimulation of intake was observed when the APJ receptor may therefore prove to be important pharmaceuti- the peptide was administered during the day-time (19). By contrast cals in the treatment of obesity. to these findings, another group reported that acute i.c.v. adminis- tration of apelin-13 had either little or no effect on food intake of rats (9). These conflicting reports could be explained as resulting Acknowledgements from differences in the species of rodent used, the form of apelin We thank Shabina Bashir for technical assistance and to the animal house used, and variation in the doses or time point of injections. More- staff at the University of Aberdeen who cared for the animals. This study over, the possible long-term effects of apelin may be more manifest was supported by Scottish Executive Environment and Rural Affairs Depart- after chronic infusion rather than bolus injection because the ment (N.H.) and the University of Aberdeen (J.R.S.). A.V. was the recipient of

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