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. 2010 Jul 7;151(9):4224–4235. doi: 10.1210/en.2010-0195

Central Injection of the Stable Somatostatin Analog ODT8-SST Induces a Somatostatin2 Receptor-Mediated Orexigenic Effect: Role of Neuropeptide Y and Opioid Signaling Pathways in Rats

Andreas Stengel 1, Tamer Coskun 1, Miriam Goebel 1, Lixin Wang 1, Libbey Craft 1, Jorge Alsina-Fernandez 1, Jean Rivier 1, Yvette Taché 1
PMCID: PMC2940496  PMID: 20610566

Abstract

Somatostatin and octreotide injected into the brain have been reported to modulate food intake. However, little is known regarding the underlying mechanisms. The stable oligosomatostatin analog, des-AA1,2,4,5,12,13-[DTrp8]-somatostatin (ODT8-SST), like somatostatin, binds to all five somatostatin receptors (sst1–5). We characterized the effects of ODT8-SST injected intracerebroventricularly (icv) on food consumption and related mechanisms of action in freely fed rats. ODT8-SST (0.3 and 1 μg per rat, icv) injected during the light or dark phase induced an early onset (within 1 h) and long-lasting (4 h) increase in food intake in nonfasted rats. By contrast, ip injection (0.3–3 mg/kg) or icv injection of selective sst1 or sst4 agonists (1 μg per rat) had no effect. The 2 h food intake response during the light phase was blocked by icv injection of a sst2 antagonist, the neuropeptide Y (NPY) Y1 receptor antagonist, BIBP-3226, and ip injection of the μ-opioid receptor antagonist, naloxone, and not associated with changes in plasma ghrelin levels. ODT8-SST (1 μg per rat, icv) stimulated gastric emptying of a solid meal which was also blocked by naloxone. The increased food intake was accompanied by a sustained increase in respiratory quotient, energy expenditure, and drinking as well as μ-opioid receptor-independent grooming behavior and hyperthermia, while ambulatory movements were not altered after ODT8-SST (1 μg per rat, icv). These data show that ODT8-SST acts primarily through brain sst2 receptors to induce a long-lasting orexigenic effect that involves the activation of Y1 and opiate-receptors, accompanied by enhanced gastric transit and energy expenditure suggesting a modulation of NPYergic and opioidergic orexigenic systems by brain sst2 receptors.

The oligosomatostatin analog ODT8-SST acts centrally to induce a long-lasting, somatostatin2 receptor-mediated, orexigenic effect dependent upon the activation of the NPY and opioid signaling system in freely fed rats accompanied by increased energy expenditure and accelerated gastric emptying.

In 1973, Guillemin and colleagues isolated somatostatin-14 (SST) from ovine hypothalami (1) and few years later, the N terminally extended form, SST-28, was characterized from porcine intestine (2). In addition to the initially established physiological role to inhibit growth hormone release from the pituitary (1), SST in the brain is known to exert multiple extrapituitary actions (3,4) through interaction with five membrane receptors, sst1-sst5 (5). Notably, several studies indicated that SST or stable analogs alter food ingestion in rats, although the data obtained were divergent. Some showed an increase in food intake (6,7,8,9,10), others a decrease (7,11,12) or a biphasic effect (13). These discrepant findings may be explained by different doses used as SST increases food intake when injected intracerebroventricularly (icv) or into the anterior piriform cortex at low doses (0.7–65 ng per rat = 0.4–40 pmol), whereas higher (3.3–4.9 μg per rat = 2–3 nmol) doses decrease chow ingestion (7,12,14).

The sst receptor subtype(s) and mechanisms through which SST injected into the brain influences food intake have not been characterized and received little attention so far. Octreotide (SMS 201–995), a stable oligosomatostatin analog, binds mainly to sst2, sst3, and sst5 receptors with highest affinity to sst2 (15,16). The peptide was reported to increase food intake when continuously infused into the third brain ventricle in ad libitum-fed rats (6). Other reports indicate that SST injected icv antagonizes the 24-h anorexigenic effect of leptin primarily through sst3/1/2 receptors (17) and partially prevents the icv corticotropin-releasing factor (CRF)-induced suppression of feeding response to a fast (18). These data were indicative of an interaction between brain somatostatin signaling and other hypothalamic neuropeptide systems regulating food intake. SST-14 and SST-28 interact with sst1–5 receptors and display nanomolar affinity to sst2 (16) including the variants sst2A and sst2B generated by alternative splicing (19). Similar to SST, des-AA1,2,4,5,12,13-[DTrp8]-somatostatin (ODT8-SST), a long-acting agonist (20) binds to all sst receptors with nanomolar affinity (21) and therefore allows to study the effects that closely resemble those exerted by the endogenous ligand. We previously reported that ODT8-SST acts in the brain to influence digestive functions indicated by the stimulation of gastric acid secretion and emptying upon intracisternal injection in rats (22,23).

In the present study, we examined the dose-related effect of ODT8-SST on food intake in rats injected icv either during the light or dark phase when the drive for food ingestion is low or already stimulated respectively (24). Because all studies related to SST or octreotide influence on food intake have been performed in rats (6,7) and recently in chicks (8), we also investigated whether mice are similarly responsive. We characterized the receptor subtype involved in ODT8-SST-induced stimulation of food intake using selective peptide sst1 or sst4 receptor agonists and a recently developed selective peptide sst2 antagonist (25,26,27). We thereafter examined whether the underlying mechanisms of ODT8-SST action involve established orexigenic pathways. Neuropeptide Y (NPY) is a major hypothalamic component mediating the signals of orexigenic peptides (28). Opioid receptors have been implicated in the central regulation of feeding (29), especially the hedonic rather than nutritional drive to ingest food, indicating a role in the rewarding aspect of eating (30), and the maintenance of NPY-induced feeding (31). Therefore, we investigated whether NPY-Y1 and μ-opioid receptor signaling pathways are involved in icv ODT8-SST orexigenic effects in the light phase using the Y1 receptor antagonist, BIBP-3226 (32) and the μ-opioid receptor antagonist, naloxone (30). Associated changes in circulating levels of acyl-ghrelin and glucose, as well as gastric emptying were assessed after icv ODT8-SST to identify possible peripheral mechanisms along with changes in ambulatory and fine movements, rectal temperature, and parameters of energy expenditure.

Materials and Methods

Animals

Adult male Sprague Dawley rats (Harlan, San Diego, CA; and Taconic, Hudson, NY) weighing 280–350 g and adult male C57BL/6 mice (23–28 g, Harlan) were group housed under controlled illumination (0600–1800 h) and temperature (21–23 C) until the start of the experiments. Animals had free access to standard rodent chow (Prolab RMH 2500; LabDiet, PMI Nutrition, Brentwood, MO) and tap water. All protocols were approved by the Institutional Animal Care and Use Committee of the Veterans Administration (99-127-07, 05-058-02) and Eli Lilly and Co. Except otherwise stated, experiments were started between 0900 and 1000 h.

Substances

ODT8-SST, MW 1078.5, compound no. 1 in (21), sst2 antagonist, des-AA1,4–6,11–13-[pNO2-Phe2,DCys3,Tyr7,DAph(Cbm)8]-SST-2Nal-NH2, MW 1208.5, compound no. 4 in (27), sst1 agonist, des-AA1,4–6,10,12,13-[DTyr2,d-Agl(NMe,2naphtoyl)8,IAmp9]-SST-Thr-NH2 MW 1238.5, compound no. 25 in (25), and the sst4 agonist, des- AA1, 2, 4, 5, 12, 13-[Aph7]-Cbm-SST, MW 1137.4, compound no. 15 in (26) (Clayton Foundation Laboratories, Salk Institute, La Jolla, CA) were synthesized and purity assessed as previously described (21,25,26,27). Peptides and the NPY-Y1 receptor antagonist, BIBP-3226 (Sigma-Aldrich, St. Louis, MO) were kept in powder form at −80 C and dissolved in pyrogen-free distilled water immediately before the experiments. Naloxone hydrochloride (Sigma-Aldrich) was dissolved in saline and the cyclooxygenase inhibitor, indomethacin (Sigma-Aldrich) in 1% sodium bicarbonate containing 10% dimethyl sulfoxide.

Intracerebroventricular injection

Procedures for implanting the guide cannula into the right lateral cerebroventricle and icv injections in 10 μl were as previously described (33). Stereotaxic coordinates were obtained from Paxinos and Watson’s brain atlas (34). After surgery, animals were housed individually, allowed to recover for 7 d, and handled daily for 5 d before the experiments. After the experiments, correct placement of the guide cannula was verified by icv injecting 10 μl of 0.1% toluidine blue.

Food intake

The dose-response effect of ODT8-SST on food intake was investigated in ad libitum-fed rats injected icv with ODT8-SST (0.1, 0.3, or 1 μg per rat) or vehicle during the light phase (0900 h) or immediately before the dark phase (1800 h). Doses of peptide were based on our previous dose-response of ODT8-SST-induced central actions to stimulate digestive functions (22,23). After injection, preweighed rat chow was made available and food intake [g per 300 g body weight (bw)] monitored at various time intervals. Based on dose-response studies, in all subsequent experiments ODT8-SST was injected icv at 1 μg per rat during the light phase. The role of brain sst2, Y1, and μ-opioid receptors in ODT8-SST orexigenic action was investigated using the sst2 antagonist (1 μg per rat, icv, 5 μl), BIBP-3226 (10 or 30 μg per rat, icv, 5 μl), naloxone (5 mg/kg bw, ip, 300 μl), or vehicle (water icv or saline ip) followed by icv ODT8-SST or vehicle (5 μl) and cumulative food intake was monitored for 2 h. The doses of the antagonists were based on preliminary data showing the blockade of the central sst2 agonist-induced orexigenic response by icv sst2 antagonist, blockade of icv NPY-induced feeding by BIBP-3226 at 30 μg per rat (32), and reduction of dark phase food intake by naloxone (35). All feeding experiments were performed in rats maintained in their familiar housing cages except a reduced amount of bedding and repeated in a crossover design. Food intake was monitored as previously (33).

Behavior

Animals were injected icv with ODT8-SST (1 μg/rat) or vehicle and placed in their home cage with free access to food and water. During the second hour after injection behaviors consisting of grooming, locomotor activity, and food (including food approach) and water intake (including water approach) were monitored manually by an observer blinded to the animals’ treatment as in our previous study (33). Each behavioral component was counted when lasting >5 sec. In a separate experiment, rats were injected ip (300 μl) with naloxone (5 mg/kg) or vehicle (saline) followed by icv ODT8-SST or vehicle and behavior was monitored during the second hour after injection. In another study, ad libitum-fed rats were placed in indirect calorimeter chambers (Oxymax; Columbus Instruments, Columbus, OH) 1 d before the experiment to get accustomed to these cages. The next day, ODT8-SST or vehicle was injected icv and ambulatory (new beam breaks per 48 min = 0.8 h) and fine movements (breaks within a beam per 48 min) were assessed for 9.6 h using the beam break method as described before (36).

To control for pica behavior, rats were injected icv with ODT8-SST or vehicle and maintained in their cages without access to food to monitor possible ingestion of bedding material for 2 h after injection.

Metabolic rate

Rats were placed in indirect calorimeter chambers (Columbus Instruments) 1 d before the experiment. The next day, ODT8-SST or vehicle was injected icv in rats with free access to food and water. Various parameters of metabolic rate were assessed as reported before (36).

Gastric emptying

Gastric emptying of ingested standard chow was determined as previously described (37). Briefly, rats were fasted for 20 h and refed starting at 0700 h for 2 h, then food and water were removed and vehicle or ODT8-SST was injected icv. Gastric emptying was assessed 2 h later. In another study, a similar protocol was used except that naloxone (5 mg/kg in 300 μl saline) or vehicle (saline) was injected ip before icv injection of ODT8-SST or vehicle.

Plasma acyl-ghrelin levels

Icv cannulated rats were equipped with a jugular vein catheter as described before (33). Rats were allowed to recover for 4 d and body weight monitored daily. Ad libitum-fed rats were injected icv with ODT8-SST or vehicle and had access to water but not food after injection. Blood (0.5 ml) was withdrawn from conscious lightly hand-restrained rats before and 1 and 3 h after injection, transferred in ice-cooled tubes containing EDTA (7.5%, 10 μl per 0.5 ml blood; Sigma-Aldrich) and aprotinin (0.6 Trypsin Inhibitory Unit/ml; ICN Pharmaceuticals, Costa Mesa, CA), and centrifuged. Plasma samples were stored at −80 C until radioimmunoassayed in a single batch for acyl-ghrelin (Linco Research, St. Charles, MO).

Blood glucose

Vehicle or ODT8-SST was injected icv, drops of blood were obtained by tail prick in conscious rats before and 20 min, 40 min, 60 min, 2 h, and 3 h after injection, and blood glucose was measured (One-Touch Ultra; LifeScan, Milpitas, CA). In another study, rats were injected with naloxone (5 mg/kg) or vehicle ip followed by icv ODT8-SST or vehicle and blood glucose measured. Ad libitum-fed rats were deprived of food but not water after injection.

See Supplemental Text, Supplemental Figs. 1–4, and Supplemental Table 1, published on the Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Statistical analysis

Data are expressed as mean ± sem and were analyzed by ANOVA followed by all pair-wise multiple comparison procedures (Tukey post hoc test) or two-way ANOVA followed by Holm-Sidak method. Time course studies were evaluated using repeated paired t tests. P < 0.05 was considered significant.

Results

Somatostatin2 receptor-mediated orexigenic action of icv ODT8-SST in rodents: role of NPY-Y1 and μ-opioid receptors

We first assessed the dose-response effect of ODT8-SST injected icv during the light or dark phase on food intake in freely fed rats. In the light phase, ODT8-SST (0.3 μg per rat = 0.31nmol) significantly increased cumulative food intake by 5.5- and 5.0-fold compared with vehicle-injected controls at 2 h and 4 h after injection, respectively (P < 0.05; Fig. 1A). Likewise, ODT8-SST (0.3 μg per rat icv) injected before lights off increased the dark phase food intake by 2.4- and 1.8-fold compared with vehicle at 2 h and 4 h, respectively (P < 0.05; Fig. 1B). ODT8-SST orexigenic effect was dose-related as a 3-fold lower dose (0.1 μg per rat, icv) had no effect during either the light or dark phase (Fig. 1), whereas a 3-fold higher dose (1 μg per rat, icv) resulted in a 42% and 39% higher cumulative light phase food intake at 2 h and 4 h, respectively, compared with 0.3 μg per rat (Fig. 1A). There was no additional increment in the dark phase food intake at 1 μg per rat, most likely related to the ceiling effect of gastric content (Fig. 1B). Therefore, the highest dose (1 μg per rat) was selected for all subsequent studies performed in rats during the light phase. When rats were injected with ODT8-SST icv and thereafter did not have access to food, no consumption of bedding material was observed (data not shown).

Figure 1.

Figure 1

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ODT8-SST injected into the lateral cerebroventricle (icv) increases light and dark phase food intake in rats. ODT8-SST (0.1, 0.3 or 1 μg per rat, icv) or vehicle was injected during the light (A) or at the beginning of the dark phase (B) in ad libitum-fed rats chronically implanted with cannula. Cumulative food intake was monitored at 2 h and 4 h after injection. Each bar represents the mean ± sem of number of rats indicated at the bottom. *, P < 0.05 and **, P < 0.01 vs. vehicle; ##, P < 0.01 vs. ODT8-SST 0.1 μg.

The time course of changes in light phase food intake after icv ODT8-SST was assessed at various intervals after injection in a separate study. There was a significant stimulation of food intake during the first hour, second hour, as well as 2- to 4-h period, whereas values were decreased between 6 and 9 h after injection (P < 0.01) compared with vehicle (Fig. 2A). Cumulative food intake over the 9-h after injection was 6.4 ± 0.7 after ODT8-SST vs. 3.5 ± 0.5 g/300 g bw after vehicle (P < 0.01; Fig. 2B). In contrast to the orexigenic effects of icv ODT8-SST, ip injection (0.3–3 mg/kg bw) did not alter food intake (see Supplemental Fig. 1A published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Figure 2.

Figure 2

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ODT8-SST injected icv increases light phase food intake in ad libitum-fed rats. ODT8-SST (1 μg per rat) or vehicle was injected icv and food intake monitored over 9 h and expressed as food intake for each period (A) or cumulative food intake (B). *, P < 0.05 and **, P < 0.01 vs. vehicle. Bars (A and B), mean ± sem of 12 animals per group.

Next, we assessed whether the ODT8-SST orexigenic effect during the light phase could be extended to mice. In ad libitum-fed mice, icv injection of ODT8-SST under brief anesthesia induced a dose-related stimulation of food intake. At 0.3 μg per mouse, the peptide increased cumulative food intake at 2 h after injection compared with vehicle (P < 0.05), whereas before or after no changes in food intake were observed (Supplemental Fig. 2). At 1 μg per mouse, ODT8-SST icv resulted in a pronounced and long-lasting raise in cumulative food intake significant at 1 h, 2 h, 4 h, and 6 h (P < 0.05) after injection, whereas the 9-h cumulative food intake was not altered (Supplemental Fig. 2). By contrast, ip injection of ODT8-SST (0.12 and 0.4 mg/kg) did not influence food intake (Supplemental Fig. 1B).

To get insight in the receptor subtype(s) involved in ODT8-SST orexigenic action during the light phase in rats, we injected selective sst receptor subtype agonists icv under similar conditions. Neither the selective sst1 agonist (1 μg per rat, icv) nor sst4 agonist (1 μg per rat, icv) resulted in significant changes in cumulative food intake (P > 0.05; Supplemental Fig. 3). However, the sst2 antagonist (1 μg per rat, icv) completely blocked the icv ODT8-SST-induced increase in cumulative food intake (Fig. 3A). We also assessed in rats whether the ODT8-SST action was involved in the established orexigenic NPY and opiate pathways (30). The NPY-Y1 receptor antagonist BIBP-3226 (10 or 30 μg per rat) icv dose-dependently inhibited the icv ODT8-SST-induced stimulation of food intake by 57% and 82%, respectively (Fig. 3B). Naloxone (5 mg/kg, ip) also completely prevented the icv ODT8-SST-induced increase in cumulative food intake (Fig. 3C). In rats injected icv with either BIBP-3226 (10 μg per rat), naloxone, or sst2 antagonist alone, food intake was similar to vehicle (Fig. 3), while after BIBP-3226 at 30 μg per rat, values were reduced compared with vehicle although the difference did not reach significance (Fig. 3).

Figure 3.

Figure 3

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A, The sst2 antagonist injected icv prevents the icv ODT8-SST-induced increased light phase food intake in ad libitum-fed rats. Vehicle or sst2 antagonist (1 μg per rat) was injected icv immediately before icv injection of vehicle or ODT8-SST (1 μg per rat) and cumulative food intake was monitored for 2 h. **, P < 0.01 vs. vehicle/vehicle, sst2 antagonist/vehicle, and sst2 antagonist/ODT8-SST. B, The NPY-Y1 receptor antagonist blocks the ODT8-SST-induced increase in light phase food intake in ad libitum-fed rats. The NPY-Y1 receptor antagonist BIBP-3226 (10 or 30 μg per rat) or vehicle was injected icv followed by icv ODT8-SST (1 μg per rat) or vehicle, and the 2-h cumulative food intake was monitored. **, P < 0.01 vs. all other groups. C, The μ-opioid receptor antagonist naloxone inhibits the icv ODT8-SST-induced increase in light phase food intake in ad libitum-fed rats. Naloxone (5 mg/kg) or vehicle was injected ip before icv injection of ODT8-SST (1 μg per rat, icv) or vehicle, and the 2-h cumulative food intake monitored. *, P < 0.05 vs. vehicle/vehicle, naloxone/vehicle, and naloxone/ODT8-SST. Bars (A–C), Mean ± sem of number of rats indicated at the bottom.

As NPY neurons are known to be activated by elevated levels of circulating ghrelin (38), we assessed whether icv ODT8-SST would influence the release of ghrelin. In rats fed ad libitum until the start of the experiment and no longer thereafter, two-way ANOVA showed a significant 96% increase in plasma acyl-ghrelin 3 h after injection of vehicle compared with preinjection values (F (2,21) = 21.7, P < 0.05; Table 1). ODT8-SST further increased plasma acyl-ghrelin by 62% at 3 h after injection compared with vehicle (P < 0.05), whereas at 1 h no changes were observed compared with vehicle (Table 1).

Table 1.

Time course of changes in plasma acyl-ghrelin levels induced by ODT8-SST (1 μg/rat) or vehicle injected icv during the light phase in conscious rats

Time (h) Plasma acyl-ghrelin (pg/ml)a
Vehicle (n = 5) ODT8-SST (n = 4)
0 71.6 ± 7.6 70.3 ± 4.6
1 120.0 ± 21.1 91.1 ± 6.8
3 140.6 ± 21.9b 228.3 ± 27.0c
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a

Data are mean±sem of number of rats in parentheses. 

b

P < 0.05 vs. vehicle at the respective time point. 

c

P < 0.05 vs. vehicle at time point 0 h (before injection). 

Central action of ODT8-SST to influence behaviors

Next, we examined whether the stimulation of food intake during the light phase was associated with other behavioral changes monitored manually during the second hour after injection. In ad libitum-fed rats, ODT8-SST icv stimulated eating (P < 0.001) and drinking behavior (P < 0.001) compared with vehicle (Fig. 4A). The peptide also increased grooming behavior (P < 0.001), whereas ambulatory activity was not altered compared with vehicle (P > 0.05; Fig. 4A). Likewise, in separate studies, ODT8-SST did not change ambulatory movements compared with vehicle as monitored automatically using the beam break method (Fig. 4B). However, fine movements mainly consisting of grooming behavior were significantly increased with a peak response at 0.8 h (P < 0.01) and a linear time-related return to values observed in the vehicle group at 4.8 h after ODT8-SST injection, while no changes were observed after vehicle icv (Fig. 4C). No abnormal behaviors known as barrel rotation, reported after SST or octreotide injected icv at doses >3 μg per rat (39,40), were observed in all our experiments. Injection of naloxone (5 mg/kg bw, ip) blocked the icv ODT8-SST-induced increase in eating (Fig. 5B) and drinking behavior (Fig. 5C), while not influencing grooming behavior (Fig. 5A).

Figure 4.

Figure 4

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ODT8-SST injected icv increases grooming behavior including scratching, washing, and licking, whereas locomotor behavior is not altered. A, Ad libitum-fed rats were injected icv with ODT8-SST (1 μg per rat) or vehicle during the light phase and behavior was monitored during the second hour after injection. **, P < 0.0.1 vs. vehicle. ODT8-SST does not alter ambulatory movements (B), whereas fine movements are significantly increased (C) as assessed by an automated beam break apparatus. *, P < 0.05 and **, P < 0.01 vs. vehicle. Arrow (B and C), time point of injection. Data in A–C are presented as mean ± sem.

Figure 5.

Figure 5

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Naloxone does not modify the icv ODT8-SST-induced grooming while blocking the eating and drinking behavior. Ad libitum-fed rats were injected icv with ODT8-SST (1 μg per rat) or vehicle during the light phase, and behaviors were monitored during the second hour after injection including grooming behavior (scratching, washing, and licking; A), eating behavior (including food approach; B), and drinking behavior (including water approach; C). *, P < 0.05 and **, P < 0.01 vs. vehicle/vehicle; #, P < 0.05 and ##, P < 0.01 vs. naloxone/vehicle; +, P < 0.05 vs. vehicle/ODT8-SST. Data in A–C are presented as mean ± sem (n = 8–10 per group).

Injection of ODT8-SST icv increases metabolic rate and rectal temperature

The enhanced orexigenic behavior induced in the light phase was accompanied by a similar time course of changes in energy expenditure (kcal/kg · h−1) with a 1.4-fold peak increase at 0.8 h (P < 0.01) and a time-related decline to reach vehicle values at 4.8 h (Fig. 6A). By contrast, the vehicle group did not show significant differences during the same time period (P > 0.05; Fig. 6A). In addition, ODT8-SST induced a rapid onset increase in the respiratory quotient (VCO2/VO2: 0.90 ± 0.03 before injection to 0.99 ± 0.02 at 0.8 h) which remained elevated for 7.2 h (P < 0.05) and gradually returned to preinjection values at 8 h after injection (Fig. 6B). This contrasts with the vehicle group which showed a time-related decrease in VCO2/VO2 from 0.92 ± 0.02 before injection to values of 0.81 ± 0.01 at 1.6 h with a nadir response at 8 h after injection (0.73 ± 0.01, P < 0.01; Fig. 6B).

Figure 6.

Figure 6

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ODT8-SST injected icv increases energy expenditure in ad libitum-fed rats. Animals were placed in calorimeter cages 1 d before the injection to get acclimated to the chambers. On the following day ODT8-SST (1 μg per rat) or vehicle was injected icv (arrow) during the light phase in ad libitum-fed chronically cannulated rats and energy expenditure (A) as well as respiratory quotient (B) measured over a period of 9.6 h after injection. Data are presented as mean ± sem of 6 rats per group. *, P < 0.05 and **, P < 0.01 vs. vehicle at the respective time point; #, P < 0.05, ##, P < 0.001 vs. same group at time point 0.

ODT8-SST icv also significantly increased rectal temperature compared with vehicle at 1 h, 2 h, and 4 h (P < 0.001; Supplemental Fig. 4A) after injection. Naloxone (5 mg/kg bw, ip) further increased the icv ODT8-SST-induced rise in rectal temperature at 2 h after injection (39.5 ± 0.2 vs. 38.5 ± 0.2 C, P < 0.05), whereas alone it had no effect on basal temperature (Supplemental Fig. 4B). Preinjection of indomethacin (10 mg/kg bw, ip) did not alter the icv ODT8-SST-induced rise in rectal temperature (Supplemental Fig. 4C).

Icv ODT8-SST-induced stimulation of gastric emptying is μ-opioid receptor-dependent

ODT8-SST icv significantly accelerated gastric emptying of an ingested solid meal compared with vehicle (P < 0.01; Fig. 7A), whereas ip injection of the peptide (30 μg/kg bw) had no effect (ODT8-SST: 63.9 ± 3.9, n = 9 vs. vehicle: 58.1 ± 6.0%, n = 8, in 2 h, P > 0.05). Naloxone (5 mg/kg bw, ip) blocked the icv ODT8-SST-induced increased gastric emptying, whereas alone had no effect (Fig. 7B).

Figure 7.

Figure 7

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A, ODT8-SST injected icv increases gastric emptying of an ingested solid meal in rats. Animals were fasted for 20 h and refed for 2 h, then food and water were removed and vehicle or ODT8-SST (1 μg per rat) was injected icv. Gastric emptying for solid food was assessed 2 h later. **, P < 0.01 vs. vehicle. B, Naloxone blocks the ODT8-SST-induced acceleration of gastric emptying. Fasted rats were refed for 2 h and naloxone (5 mg/kg) or vehicle were injected ip followed by ODT8-SST (1 μg per rat) or vehicle icv and gastric emptying was measured 2 h later. **, P < 0.01 vs. vehicle/vehicle. Bars (A and B), mean ± sem of number of rats indicated at the bottom.

Changes in circulating levels of glucose are reflected in brain extracellular glucose levels (41) which may influence food intake and gastric motility when large variations occur (42,43). Therefore, we assessed glycemia after ODT8-SST in rats fed ad libitum until the start of the experiment. Before injection both groups had similar glycemia values (Supplemental Table 1). Upon vehicle icv, blood glucose rose from 108.0 ± 2.2 to 116.9 ± 4.0 mg/dl at 20 min and remained within the 116–117 mg/dl range throughout the 3-h experimental period (P < 0.05 at 3 h vs. pre-injection). After ODT8-SST icv, blood glucose was not significantly different from preinjection values for the 3-h period and was significantly lower at 2 h and 3 h after injection compared with vehicle (P < 0.01; Supplemental Table 1). Injection of naloxone (5 mg/kg bw, ip) did not modify the blood glucose values after ODT8-SST icv at 2 h after injection (naloxone+ODT8-SST: 102.4 ± 5.7, n = 6 vs. vehicle+ODT8-SST 101.4 ± 2.0 mg/dl, n = 8, P > 0.05).

Discussion

In the present study, we provide evidence that the oligosomatostatin agonist ODT8-SST injected icv during the light phase increases food intake, fine movements including grooming behavior, energy expenditure, rectal temperature, and gastric emptying in rats fed ad libitum. These effects are centrally mediated because ip injection of ODT8-SST did not alter food intake, behavior, or gastric emptying, suggesting that activation of brain somatostatin signaling pathways coordinates stimulation of food intake and digestive motor function.

ODT8-SST injected acutely into the lateral cerebroventricle in chronically cannulated rats resulted in a dose-dependent (0.1–1 μg per rat) food consumption in ad libitum-fed rats. The orexigenic effect was not restricted to the light phase when the drive to eat is low and also observed during the nocturnal phase under conditions of a physiological drive to eat (24). The heightened nocturnal food intake indicates an additive effect of the somatostatin analog to already activated orexigenic mechanisms including the hypothalamic NPY and opioid signaling system (44,45). We ruled out that the ODT8-SST action reflects pica behavior as bedding material was not consumed in the absence of food. In addition, the orexigenic property of ODT8-SST was not restricted to one species as a dose-related stimulation of light phase food intake was reproduced in freely fed mice after acute icv injection. The orexigenic effect induced by icv ODT8-SST was centrally mediated in rats and mice as the peptide did not influence food ingestion when injected ip at 1000- or 10-fold higher doses respectively compared with the highest effective icv dose. The peptide action was rapid in onset and long-lasting, shown by the significant increase in food intake occurring during the first hour and maintained during the second hour and 2- to 4-h periods after injection. The long-lasting response differs from that of other orexigenic peptides (e.g. ghrelin or galanin) reported to induce a short-lasting stimulation of food intake after icv injection (46,47). Although it can reflect a property of the stable analog, there is evidence that icv injected somatostatin-14 increases meal frequency and counteracts the leptin-induced decrease in food consumption when monitored 24 h after peptide injection (17), indicative of a long-term action of the native peptide as well.

The robust orexigenic effect of ODT8-SST is likely to be sst2 receptor-mediated because icv injection of the sst2 antagonist blocked the 2-h increase in cumulative food intake. In addition, icv injection of selective sst1 or sst4 agonist did not reproduce the response induced by icv ODT8-SST, arguing against a role of these receptor subtypes. A previous report showed that octreotide, displaying high affinity to sst2, moderate affinity to sst3 and sst5, and no affinity to sst1 and sst4 receptors (48), injected chronically icv enhances food intake in rats (6). There is also evidence that the sst2 analog, L-779 976 injected icv increases the 24-h meal frequency in rats (17). Somatostatin receptors have been described in various regions of the brain and, although overlap exists, the distribution pattern of sst receptor subtypes is distinct (49,50). The sst2A receptor expression at the gene and protein levels have been described inter alia in nuclei implicated in food intake regulation including the supraoptic nucleus, hypothalamic paraventricular and arcuate nucleus, and ventromedial and lateral hypothalamus in rats and mice (49,50,51,52,53). This provides an anatomical substrate for the sst2 receptor-mediated orexigenic response that needs to be further localized. Collectively, these data support that activation of the brain sst2 receptor signaling pathway is primarily involved in mediating the orexigenic action of ODT8-SST. In support of a physiological role of brain SST in the stimulation of food intake are previous reports showing that chronic third ventricular infusion of SST antiserum over 2 d results in decreased daily cumulative food intake (6) and that hypothalamic somatostatin content displays circadian variations with a peak at the beginning of the dark phase when rats show their maximal food consumption and lowest levels in the early light phase (54). However, whether sst2 receptors may be part of the circuitries involved in the nocturnal drive to eat remains to be investigated.

The underlying brain mechanisms through which ODT8-SST stimulates light phase food intake during the first hour after icv injection is unlikely to be related to a surge in ghrelin release. We did not find a significant change in plasma ghrelin compared with vehicle at 1 h after injection. However, icv ODT8-SST increased plasma acyl-ghrelin at 3 h after injection, which may contribute to maintain eating behavior during this time. The observed increase of acyl-ghrelin at 3 h after icv injection of vehicle may reflect a fasting-associated rise in acyl-ghrelin since the animals were food deprived after injection. Likewise, we can rule out that changes in glycemia underlie ODT8-SST’s orexigenic effect. Blood glucose was not significantly changed during the 3 h after ODT8-SST injection compared with preinjection values. However, ODT8-SST blocked the 9% rise in glycemia occurring in the vehicle group at 3 h after injection most likely linked with repeated tail prick blood samplings in conscious rats. This is consistent with earlier reports showing that icv ODT8-SST acts in the brain to reduce stress-related increases in blood glucose in rats (55). Additionally, this relative decrease is most likely not sufficient to increase food intake by itself based on evidence that only pronounced hypoglycemia (<60 mg/dl) [e.g. by injection of insulin] robustly increases food intake in rats (42).

In the present study, icv injection of the selective NPY-Y1 receptor antagonist BIBP-3226 dose-dependently reduced the ODT8-SST-induced stimulation of food intake in ad libitum-fed rats suggesting that the mechanisms underlying the ODT8-SST orexigenic effect could involve activation of hypothalamic NPY-Y1 pathways. Using similar doses, BIBP-3226 at 30 μg per rat icv was previously reported to abolish the NPY-induced food intake (32), whereas a lower dose (5 μg per rat, icv) only blunted the peptide’s orexigenic action (56). Involvement of the NPY signaling system is also supported by the observation that SST derived from the periventricular nucleus (53) could excite sst2 receptor-bearing NPY neurons in the arcuate nucleus (57). In addition, in line with an established relationship between NPYergic and opioidergic systems in the regulation of appetite (58), the μ-opioid receptor antagonist naloxone injected peripherally also abolished ODT8-SST’s orexigenic effect. Both naloxone and BIBP-3226 did not significantly influence light phase food intake of ad libitum-fed rats when injected alone as shown in previous studies (32,59), most likely because of the low endogenous drive to eat during that time (24). Therefore, the present data suggest that icv ODT8-SST may exert its stimulatory effect through the NPY→opioid pathway, established to regulate feeding (30). Because ODT8-SST has a strong orexigenic effect as reflected by the sustained stimulation of food intake not only in the light but also in the dark phase, a rewarding component mediated by central μ-receptor opioidergic signaling would be consistent with the recruitment of such mechanisms. Moreover, at the cellular level, sst receptors have been established to form not only homodimers but also heterodimers with opioid receptors (60), although occurrence and functional implications in vivo will have to be further assessed. Additionally, it cannot be ruled out that other peptidergic circuitries and transmitters involved in the brain regulation of feeding behavior (61) may also contribute to ODT8-SST food intake stimulatory actions.

Besides influencing food intake, central injection of SST modulates other behaviors. However, there are differential effects in relation to the dose. Whereas low doses (<1 μg per rat) activate sniffing and exploratory behavior, higher doses (1–10 μg per rat) shortly increase those behavioral parameters followed by a decrease (62). In the present study, ODT8-SST injected at 1 μg per rat icv increases grooming, eating, and drinking behavior during the second hour after injection, while not altering locomotion as monitored visually. Likewise, automated assessment showed an increase in fine movements mainly representing grooming behavior, whereas ambulatory movements representing locomotor activity were unchanged. These findings are in accordance with a study showing increased grooming behavior following ODT8-SST administration (0.3–3 μg per rat, icv) (63). The increase in grooming behavior along with the stimulation of food ingestion reproduces features of the dark phase when nocturnally feeding rats consume the majority of their daily food intake (24). As somatostatin was reported to be involved in the entrainment of circadian clocks in the suprachiasmatic nucleus (64), this action may play a role in the observed behavioral changes that needs to be explored. In the present study the μ-opioid receptor antagonist naloxone selectively blocked ODT8-SST’s effect on eating and drinking behaviors, whereas grooming was not altered. Other studies addressing the dipsogenic effect of icv octreotide showed a μ-opioid receptor-independent mechanism (65). Therefore it is likely that the increased drinking behavior observed in the present study is secondary to the concomitant μ-opioid receptor-dependent increase in food intake rather than representing a direct thirst-related effect. Taken together, these data provide evidence that ODT8-SST induces ingestive behaviors (eating and drinking) via an opioid-dependent pathway, whereas the effect on grooming can be dissociated and does not involve opioidergic downstream signaling. The blockade of central opioid receptors was previously reported to abolish stress-induced food intake (66). As stress stimulates central somatostatin release (67), activation of sst2 receptors may also contribute to the opioid-dependent mechanisms of stress-related alterations of food intake that remains to be investigated.

Increased movements and food intake can be associated with changes in energy expenditure. Indeed, as measured by indirect calorimetry, ODT8-SST injected in the light phase increased energy expenditure over a period of 4 h. Consistent with these data, previous studies showed that ODT8-SST injected icv at a similar dose induced an increase in oxygen consumption within 45 min that remained elevated for the 90 min duration of the experiment (68). Moreover, we found that ODT8-SST induced a rapid onset and sustained increase in the respiratory quotient reflecting preferential glucose oxidation and less lipid oxidation over the 7 h after injection, while the stimulation of hourly food intake was no longer observed after 4 h after injection. Similarly, the orexigenic melanin-concentrating hormone (MCH) was shown to increase glucose oxidation while decreasing lipid oxidation which was associated with hyperphagia and increased energy expenditure in freely fed rats (69). Likewise, blockade of central glucagon-like peptide (GLP)-1 signaling using a GLP-1 antagonist increased food intake, respiratory quotient, and energy expenditure in mice (70). Food intake itself is known to increase glucose oxidation and decrease lipid oxidation (71). However, icv ODT8-SST also increased VO2 when rats did not have access to food and water (68). Therefore, the ODT8-SST-induced increase in respiratory quotient is unlikely to be secondary to the orexigenic effect.

We could furthermore show that ODT8-SST injected icv induces a long-lasting increase in rectal temperature by 2 C reaching a peak response of 38.5 C. Naloxone did not block the hyperthermia induced by icv ODT8-SST as reported for the hyperthermic response to icv SST-28 (72) and further increased the thermogenic response as observed previously under conditions of hyperthermia in rats kept at varying ambient temperatures (73). These data suggest that opioid-dependent caloric intake during the light phase is not related to increased thermogenesis which is opioid receptor-independent. In the present study, indomethacin did not modulate the hyperthermia after ODT8-SST, indicating a prostaglandin-independent pathway which is in agreement with a previous study of Brown and colleagues (74). The marked drop in body temperature following depletion of endogenous somatostatin by cysteamine (75) gives support for a physiological role of somatostatin in the regulation of body temperature.

Centrally acting transmitters that stimulate food intake often also accelerate gastric emptying as shown for, e.g. TSH-releasing hormone (76) or ghrelin (77). Here we show that ODT8-SST icv accelerates gastric emptying of a solid meal ingested before peptide administration. We previously reported that ODT8-SST injected into the brainstem at the level of the cisterna magna under short isoflurane anesthesia stimulates gastric emptying of a nonnutrient viscous solution (23) and gastric acid secretion (78). Collectively, these data are indicative of a coordinated central action of ODT8-SST to promote food intake and associated digestive processes. This is further supported by similar mechanisms involved in the stimulation of gastric emptying and food intake which are both blocked by pretreatment with naloxone. However, it is well established that activation of opioid receptors in the brain inhibits gastric emptying (79), whereas central NPY through Y1 signaling stimulates gastric motility (80). Therefore, ODT8-SST could increase gastric emptying via the recruitment of NPY-Y1 signaling as observed for the stimulation of food intake. Naloxone may interfere with the NPY-Y1-mediated action as demonstrated for the central NPY-induced increase in respiratory quotient that is dampened by naloxone (81).

In summary, these data show that ODT8-SST injected icv at low doses in the light phase acts in the brain to induce a long-lasting stimulation of food intake in freely fed rats associated with accelerated gastric emptying. The peptide’s orexigenic effect is mediated primarily through activation of sst2 receptors and is reproduced in mice. It is not restricted to the light phase and also occurred under already endogenously stimulated feeding conditions during the dark phase. The underlying mechanisms of ODT8-SST orexigenic response are linked with the recruitment of NPY-Y1-opioid signaling systems involved in the initiation and rewarding aspects of feeding and not associated with changes in circulating acyl-ghrelin or glucose. In addition, icv injection of ODT8-SST increased grooming behavior and rectal temperature through opiate-independent mechanisms indicating the recruitment of separate neural networks for ODT8-SST effects on feeding vs. grooming behavior/thermogenesis. The hyperthermic response was most likely caused by an increase in energy expenditure. Taken together, these data provide novel insight into central mechanisms involved in the orexigenic action of SST using a stable oligosomatostatin receptor agonist that could be a useful tool to study the central regulation of food intake by sst receptors.

Supplementary Material

[Supplemental Data]
en.2010-0195_index.html (905B, html)

Acknowledgments

We are grateful to Honghui Liang and Hsui-Chiung Yang for their excellent technical support, and we thank Eugenia Hu for reviewing the manuscript.

Footnotes

This work was supported by German Research Foundation Grants STE 1765/1-1 (to A.S.) and GO 1718/1-1 (to M.G.), National Institutes of Health (NIH) Grant R01 DK-33061, NIH Center Grant DK-41301 (Animal Core), and Veterans Affairs Research Career Scientist award (to Y.T.). J.R. is the Dr. Frederik Paulsen Chair in Neurosciences Professor.

Disclosure Summary: A.S., M.G., L.W., and Y.T. have nothing to declare. T.C., L.C., and J.A.-F. are employed at Eli Lilly and Co. J.R. is Founder of Sentia Medical Sciences, Inc. No conflicts of interest exist.

First Published Online July 7, 2010

Abbreviations: bw, Body weight; icv, intracerebroventricularly; NPY, neuropeptide Y; OD8-SST, des-AA1,2,4,5,12,13-[DTrp8]-somatostatin; SST, somatostatin.

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