To investigate a role for ghrelin in the rewarding properties of HFD, we adapted a version of the CPP task which is typically used in drug abuse studies. In this task, mice were conditioned to associate one chamber of the CPP apparatus with HFD and a 2nd chamber with an equal calorie amount of regular chow (RC). After a 12 day conditioning period (6 days on the HFD-paired side alternating every-other-day with 6 days on the RC-paired side), mice were permitted free access to both chambers in the absence of food. CPP scores were calculated by subtracting the time spent in the RC-paired chamber from the time spent in the HFD-paired chamber. Place preference for HFD was demonstrated by a positive CPP score, meaning that the mice preferred to spend time in the chamber that they associate with the more rewarding food. To ascertain whether ghrelin modulates CPP for HFD, ad lib-fed wild-type mice were injected with ghrelin (2 μg/g BW) or saline 20 min prior to placement in the CPP apparatus on the test day. Saline-treated mice spent a similar amount of time in each chamber after the conditioning period (on the test day) as before the conditioning period (on the pretest day; , n=29). In contrast, mice treated with ghrelin prior to testing spent significantly more time in the HFD-paired chamber after the conditioning period (, n=15). This finding suggests that rises in ghrelin are sufficient to induce expression of CPP for HFD or may act like a cue used in the expression of CPP for HFD. Next, mice were treated with ghrelin (2 μg/g BW) prior to each conditioning session, but not on the test day. Mice treated this way displayed a clear preference for the HFD-paired chamber (, n=22), indicating that rises in ghrelin also enable acquisition of CPP for HFD. To confirm the specificity of our findings, we repeated the experiment using GHSR-null mice (). Saline-treated GHSR-null mice spent a similar amount of time in each chamber after and before the conditioning period (n=20). In contrast to wild-type mice, GHSR-null mice did not elicit CPP for HFD upon administration of ghrelin (2 μg/g BW) either prior to testing (n=16) or prior to each conditioning session (n=16).
Figure 1 Ghrelin affects the rewarding value of HFD in a food CPP task. A. Administration of ghrelin (2 μg/g BW, s.c.) to ad lib-fed wild-type mice either acutely before the test or before each conditioning session increases the preference for the HFD-paired (more ...)
It is well known that ghrelin is physiologically increased upon calorie restriction, and that in this situation there is an enhancement in food CPP performance (30
). To determine whether the enhanced food CPP performance associated with calorie restriction is due to the elevated levels of endogenous ghrelin associated with calorie restriction, we performed CPP experiments using a recently reported GHSR antagonist, Compound 26 (27
). Calorie-restricted mice were provided ad lib
access to RC in their home cages between 12:00 p.m. and 4:00 p.m. Mice required one week to adjust their food intake amount to this 4-h calorie restriction experimental paradigm. Thus, during the first week of calorie restriction, mice gradually increased and then stabilized their daily intake of RC during the 4 h that food was available, as follows: 1.36±0.10, 1.55±0.18, 1.84±0.13, 2.13±0.07, 2.11±0.09, 2.27±0.12, and 2.42±0.13 g of RC. Behavioral studies on calorie restricted mice were started following this initial adjustment week. Throughout the remaining days of the experiment, the mice ate an average of 2.87±0.15 g of RC (nearly 8.0 kcal/day), which represents approximately 70% of the daily food intake of ad lib
-fed wild type mice in our experimental condition (4.09±0.14 g or nearly 11.9 kcal of RC). After the initial adjustment week, calorie restricted mice maintained body weights that were about 85% the average body weights of the ad lib
-fed wild- type mice. Food CPP tasks were started after the initial adjustment week and were performed just prior to the 4 h period of food availability, when plasma acylated ghrelin levels were found to be increased two-fold (13.2±1.0 vs. 5.8±0.6 pg/mL in calorie-restricted vs. ad lib
-fed mice, p<0.01). Compound 26 (30 μg/g BW) was administered either 1 h before the test or 1 h before each conditioning session to calorie-restricted mice. As expected, calorie-restricted mice treated with vehicle spent more time in the HFD-paired chamber on the test day as compared to the pretest day (, n=34). Calorie-restricted mice treated with Compound 26 only on the test day also displayed a higher mean test CPP score than pretest CPP score (, n=10). In contrast, Compound 26 given during the conditioning period completely blocked the acquisition of food CPP in calorie-restricted mice (, n=14). Thus, the endogenous rise in ghrelin that occur in the setting of calorie restriction is required for acquisition but not expression of CPP for HFD.
Figure 2 Ghrelin signaling is required for the enhancement in food CPP performance induced by calorie restriction. A. The GHSR antagonist Compound 26 (30 μg/g BW, via gavage) blocks the acquisition of food CPP in calorie-restricted mice when used before (more ...)
To corroborate our findings with Compound 26, we also performed CPP experiments using GHSR-null mice. For these experiments, GHSR-null and wild-type littermates were calorie restricted during the week preceding conditioning and throughout the pretest, conditioning and test days. Instead of the 4-h calorie restriction paradigm used above, here animals were provided access to an amount of RC corresponding to 80% of their average daily food intake [an average of 3.31±0.21 g of RC (nearly 9.5 kcal/day)]. Both groups of 80% calorie restricted mice maintained body weights that were about 95% the average body weights of ad lib
-fed mice. Food CPP tasks were performed just prior to the feeding period. Using this 80% calorie restriction protocol, wild-type mice spent more time in the HFD-paired chamber on the test day as compared to the pretest day (, n=22). In contrast, GHSR-null mice failed to show a calorie restriction-induced CPP (, n=22). Thus, both genetic and pharmacologic interference with endogenous ghrelin signaling have the ability to block CPP to HFD. Of note, different results were observed with the GHSR-null mice when the more food-limiting 4-h calorie restriction paradigm was used instead of the 80% calorie restriction. Under the 4-h protocol, both wild-type mice and GHSR-null littermates demonstrated CPP for HFD (Fig. S3
It is well established that following periods of calorie restriction, mice respond with a homeostatic drive to replenish energy stores through hyperphagia. Interestingly, in our experiments described above, Compound 26 administration to calorie-restricted mice only on the test day or only during the conditioning period both failed to block the compensatory hyperphagia observed at the end of the test when the mice were given free access to RC (). Similarly, 80% calorie-restricted wild-type mice and GHSR-null littermates both experienced compensatory hyperphagia at the end of the test when the mice were given free access to RC (). As such, while calorie restriction-associated increases in endogenous ghrelin are required to demonstrate CPP for HFD (a measure of food incentive value), ghrelin signaling does not appear to be required for the compensatory hyperphagia (a measure of homeostatic drive) that normally follows chronic calorie restriction.
Figure 3 Ghrelin signaling is not required for the compensatory hyperphagia that follows chronic calorie restriction. A. Compound 26 administered before either the test session or each conditioning session fails to block calorie restriction-associated overeating (more ...)
As previous work indicates that orexin neurons become activated by cues associated with different rewards, including addictive drugs and food (32
), we next investigated whether orexin neurons contribute to ghrelin's enhancement of CPP for HFD. We assessed c-fos expression, a marker of neuronal stimulation, within orexin neurons in mice given either saline or ghrelin prior to each conditioning session but not prior to testing. For this procedure, brains were collected from mice 2 h following their test day exposure to the CPP apparatus. Mice that exhibited ghrelin-induced acquisition of food CPP showed increased c-fos expression within LHA orexin neurons. Quantitative analysis indicated that 29.0 ± 5.2% of orexin-positive neurons were positive for c-fos in the LHAs of saline-treated mice, while in the LHAs of ghrelin-treated mice, 43.2 ± 2.3% of orexin-positive neurons were positive for c-fos (p<0.05 vs. saline-treated mice, ). The distribution of the orexin-positive neurons positive for c-fos in the LHA did not show any particular topography within the LHA.
Figure 4 Ghrelin-induced acquisition of food CPP activates orexin neurons in the lateral hypothalamus. Representative photomicrographs of brains processed by dual-label immunohistochemistry for orexin (red) and c-fos (brown) (A) and quantification of dual-labeled (more ...)
Next, we determined if orexin signaling is required for the ghrelin-induced acquisition of food CPP. Administration of the orexin receptor 1 selective antagonist SB-334867 (10 μg/g BW) to mice subsequently receiving ghrelin before each conditioning session blocked the acquisition of food CPP () while pretreatment with SB-334867 alone (without subsequent ghrelin) did not affect the performance in the food CPP protocol (Fig. S4
). This dose of SB-334867 was shown to have no anorexigenic (or orexigenic) effect on intake of freely-available food (data not shown). In addition, orexin-deficient mice (25
) failed to show ghrelin-induced acquisition of food CPP (). SB-334867-pretreated mice and orexin-deficient mice both displayed full orexigenic responses to ghrelin (for both HFD and RC) as measured during the conditioning phase of the CPP trial (). Thus, orexin pathways facilitate the ghrelin-induced acquisition of food CPP, however, ghrelin-induced stimulation of intake of freely-available food seems to be independent of orexin signaling under these particular experimental conditions.
Figure 5 Ghrelin's actions on food CPP require intact orexin signaling. A. Orexin 1 receptor antagonist (SB-334867, 10 μg/g BW, i.p.)-pretreated mice and orexin-deficient mice fail to demonstrate ghrelin-induced acquisition of food CPP. B. SB-334867-pretreated (more ...)
Finally, we tested mice using an operant conditioning protocol under a progressive ratio schedule (33
). In this measure of food motivation, the number of nose pokes required to obtain each successive reinforcing HFD pellet increases by progressive increments. The session continues until a 10 min period elapses during which the mouse does not obtain a HFD pellet. The last progressive ratio successfully completed before the end of the session is defined as the breakpoint. Ad lib
-fed mice receiving ghrelin (2 μg/g BW) 10 min prior to placement into the operant chamber showed a higher breakpoint as compared to saline-treated mice (, n=19). Pretreatment of ad lib
-fed mice with SB-334867 (10 μg/g BW) blocked the ghrelin-induced increase in breakpoint (, n=16), without altering the performance of mice injected with saline. In addition, orexin-deficient mice treated with ghrelin also failed to show a significant increase of the breakpoint (, n=5). These results confirm that ghrelin increases the motivation to obtain HFD pellets, and that this behavior requires intact orexin signaling.
Figure 6 Ghrelin increases the motivation to obtain HFD pellets in an orexin-dependent manner. Ghrelin increases the breakpoint for HFD pellets in an operant conditioning trial using a progressive ratio schedule. In contrast, SB-334867-pretreated (10 μg/g (more ...)