Effects of chronic scheduled–caloric dietary on ghrelin secretion and food intake in adult male rats

Introduction: Effects of chronic scheduled dietary on plasma ghrelin and food intake in adult male rats were assessed. Methods: Forty male Wistar rats (180-200g) were distributed into four groups (n=10), freely fed rats (control) and three scheduled-fed groups with different caloric intakes: high fat, standard and restricted diet. Then, plasma ghrelin and food intake were measured on days 0, 7 and 14. Results: Plasma ghrelin was significantly different among all groups, the scheduledstandard rats having the lowest ghrelin on day 7 and the restricted rats having the highest ghrelin on day 14. Noteworthy, fasted ghrelin in controls was as much as that of schedule groups exception restricted diet at the end of experiment. Controls consumed stable food over the time, while schedule groups showed time and caloricdependency of food intake. Schedule-standard and restricted groups on feeding time consumed high level of food. Schedule-high fat group displayed a time dependent reduction in food intake. A positive correlation was found between plasma ghrelin and food intake in fasting status for freely fed rats and anticipating status for standard and restricted-schedule groups. Conclusion: A component of ghrelin secretion can be entrained or learned for time feeding as well as long last-fasting and more is affected by quantitative and qualitative of caloric intake. Elevated ghrelin levels in restricted model are subjective both by low energy levels and learning. Also, caloric intake amount can be controlled by learning; we observed a reduction in meal size in scheduled–high fat diet. iD D ow nl oa de d fr om p pj . p hy ph a. ir at 1 0: 11 + 03 30 o n M on da y N ov em be r 4t h 20 19 chronic scheduled–caloric dietary Physiol Pharmacol 23 (2019) 189-196 | 190 (Perello et al., 2015; Wren et al., 2001; Faulconbridge et al., 2003). Ghrelin is commonly referred to as a “hunger” hormone and food deprivation index that stimulates hunger and initiates meals. Although less is known about mechanisms underlying ghrelin regulation of feeding behavior, the hypothalamic arcuate nucleus (ARC) appears to play a critical role in the ghrelin effects on food intake and satiety (Lee et al., 2016). The ARC contains different groups of neurons such as neuropeptide Y (NPY)/ agoutirelated protein (AgRP) neurons and proopiomelanocortin (POMC)/ amphetamine-regulated transcript (CART) neurons whose activity is differentially affected by ghrelin, such that ghrelin enhances the activity of NPY and AgRP neurons while suppressing the activity of POMC/CART neurons (Cowley et al., 2003). The effects of ghrelin on the activity of ARC neurons are critical for eating behavior, since ghrelin is unable to enhance food intake in mice lacking NPY/AgRP neurons (Chen et al., 2004). Ghrelin levels are flexible to changes in macronutrient composition and/or the type of diet of that is being consumed, such that modified sham feeding of different types of diet (e.g. high-fat, carbohydrate or protein) all induced a ghrelin response; however, there was a markedly greater increase in circulating ghrelin concentrations with high-protein foods (Zhu et al., 2014). In addition to the regulation of ghrelin levels by nutrient content, a growing body of literature in both human and animal models indicate that ghrelin is more appropriately described as a signal for the anticipation of food intake. For example, animals that learn to anticipate meals when placed on a meal entrainment schedule exhibit increasing levels of circulating ghrelin prior to the ingestion of the meal (Drazen et al., 2006; Hsu et al., 2016). Similarly, a robust increase in ghrelin levels immediately before each scheduled meal (breakfast, lunch and dinner) is demonstrated in humans with meals provided on a fixed schedule throughout a 24hour period (Frecka et al., 2008; Verbaeys et al., 2011). Human studies have also demonstrated that varying habitual feeding times (e.g., short or long inter-meal intervals) potently influences temporal patterns of circulating ghrelin levels, where peak ghrelin concentrations occur immediately prior to an individual's habitual meal time. These results suggest that ghrelin is not exclusively a “hunger signal” whose release is just affected by the magnitude of food deprivation, but ghrelin secretion can elevate in anticipation of food intake based on learned and habitual feeding patterns. However, whether palatability of the diet (for example high caloric content), habitual feeding patterns and the magnitude of food restriction modulate ghrelin release independently of each other, require further investigation. Changes induced in the ghrelin system potentially contribute to the inter-individual differences in body fat mass and thereby may play an important role in the obesity development. By modulating the response of ghrelin secretion before each meal, we may be able to minimize the effects of eating large meals on weight gain. Therefore, we designed the present study to test the hypothesis that a combination of anticipation of a meal and type of caloric intake could differentially affect ghrelin secretion response compared with longlast fasting. Materials and methods All experimental protocols used in this study were approved by the Ethical Committee of Isfahan University of Medical Sciences (Isfahan, Iran) in accordance with the Guide for Care and Use of Laboratory Animals (National Institute of Health Publication No.80-23, revised 1996). Forty male Wistar rats (180-200g) were housed in 12:12-h lighting cycle with ad libitum access to food and water and 23±2°C ambient temperature and 45–65% humidity (n=10). Rat entrained to a 12:12 DL cycle (reverse cycle) for a week prior to further operations. In reverse cycle enabled us to provide access to food during specific times of dark phase. Experimental procedure Rats were assigned to one of four weight-matched groups: a freely-fed group with 24h access to standard chow and 3 schedule-fed groups that were given access to the food 3 times during the dark phase aligned to their active phase with 3 different calorie intake: high fat, standard and restricted diet. In a high-fat diet, cholesterol (35% fat from lard, 60% kcal from fat) was provided into 3 meals (Gajda et al., 2008). While in the standard diet, amount of standard chow was free just at 3 meals for rats and in the restricted group, the food was well-defined and restricted (4g/time/rat; 60%) for animals (Suckow D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 11 + 03 30 o n M on da y N ov em be r 4t h 20 19 191 | Physiol Pharmacol 23 (2019) 189-196 Halabian et al. et al., 2005). Rats were maintained on either diet for 14d. Food consumption and body weight was measured on days 0, 7 and 14 in 12-14 fasted condition when they were free to food access for 1 hour (group of freely-fed rats was food deprived for the same amount of time as meal-fed rats before their usual meal, dark phase). For determination of hormone secretion output, tail blood sampling technique was used to collect blood (at the amount of 500μl) from the rats around 9 am on days 0, 7 and 14 (group of freely-fed rats was food deprived for the same amount of time as meal-fed rats before their usual meal, dark phase). Plasma acyl ghrelin was measured using rat ghrelin EISA kit with intra-assay precision CV< 10% (Zellbio Company ELISA kitGermany). Statistical analysis Results are expressed as mean±SEM. Statistical analysis was performed using SPSS 16 (SPSS Inc., Chicago, IL, USA). Statistical evaluation was carried out using a one-way analysis of variance, followed by Tukey test for comparison of means. Correlation between changes in ghrelin level and food intake was performed using Pearson correlation at the 5% error term. The P<0.05 was considered statistically significant.