Ghrelin, orexin, and galanin are orexigenic factors in rodents and humans which participate in adaptive response to weight loss. On the other hand, weight loss and fasting is accompanied by increased levels of epinephrine (Ep) and cortisol (Cor). In this study, we investigated the effects of Ep and Cor on fasting-induced ghrelin, orexin, and galanin secretion in rats fed different levels of their energy requirements.
Forty five male Wistar rats (300-350 g, 15 per group) were fed a diet containing 100, 50, and 25% of their energy requirement for 10 days followed by 2 days of fasting. Animals were then anesthetized for carotid artery cannulation for injections and blood samplings.
Rats received either 3 µg Ep/kg body weight (BW), 3 µg Cor/kg BW, or a combination of those two (0.1 mg in 1 ml of phosphate-buffered saline). Blood samples were collected before, 30, 60, and 120 min after injection.
In normal and 50% food restricted groups, fasting ghrelin levels fell after Ep and combination of Ep and Cor injection (P ≤ 0.05), whereas, orexin were decreased by combination of Ep and Cor injection in rats fed 100% of their needs and Ep alone in rats fed 50%. Galanin just fell after combination of Ep and Cor injection in both starved (50%) and normal rats. In contrast, all groups whit 25% fed ad libitum did not response to any injections (P > 0.05).
These results indicate that Ep suppresses starvation-induced secretion of ghrelin, orexin, and galanin in normal (100%) and starved (50%) rats and their response to Ep might be affected by weight loss.
Ghrelin, orexin, and galanin are neurohormones involving in food intake and energy homeostasis (1,2,3,4,5). Their orexigenic role is well understood (5,6,7). Moreover, they play an important role in short- and long-term energy balance and ghrelin and orexin are adversely related to BMI (3,8).
Although, role of ghrelin, orexin, and galanin as orexigenic hormones in appetite axis and energy balance is well known but, the effect of other factors on their secretion is still unknown.
Stress is a factor that could have different effects on appetite axis and energy homeostasis in acute and chronic stressful condition (9,10,11). Under calorie restriction, stress is high which is accompanied with increased level of cortisol (Cor) and epinephrine (Ep) (12). To study whether calorie restriction induced change in energy balance involving hormone secretory pattern could be the consequence of stress, we examined Cor and Ep intracarotid infusion on ghrelin, orexin, and galanin secretion in rats submitted to a calorie restriction equivalent to 100, 50, and 25% of usual intake for 10 days. In addition, in this study, we analyzed role of glucose as mediator in this process.
Material and Method
Adult male Wistar rats (300-350 g) were maintained under controlled temperature (20ºC) and lighting (1200-1200), with ad libitum access to standard rodent chow and tap water.
Cor and Ep were dissolved in phosphate-buffered saline (0.1 mg in 1ml phosphate-buffered saline) and stored in 4-8ºC.
Rats were divided in three groups, were kept in individual cages and fed daily a diet containing 100, 50, and 25% of their energy requirement but had free access to tap water for 10 days in three diet groups. All experimental protocols were conducted at same time (08:00 in the morning) of the day in rats fasted for 48 h after diet procedure. The left carotid was cannulated in fasted rats anesthetized with a mixture of Ketamine (75 mg/kg i.p.) and Xylazine (5 mg/kg i.p.). The catheter was opened and blood (0.5 ml) was withdrawn into a syringe before injection. Rats in each diet group were divided in three subgroups and received (3 µg/kg body weight (BW)) Ep, Cor and combination of both through intracarotid catheter for 1 min. Blood sampling were done 30, 60, and 120 min after injection. Blood was collected in Eppendorf tubes containing Heparin (2 uint/1ml blood). Plasma was separated by centrifuging (3500 rpm) for 15 min. Plasma was kept at −80ºC until assay.
Hormone assays and Glucose Measurements
Plasma levels of total ghrelin, orexin, galanin, and ACTH, Ep and corticosterone was measured by a ghrelin, orexin, galanin, (rats/mouse) radioimmunoassay kit (Milipore, Billerica, MA), ACTH and Ep (Abnova, Taipei, Taiwan) and corticosterone (rats/mouse) enzyme-linked immunosorbent assay kit (Demeditec, Kiel, Germany). Blood glucose was measured using a commercially available glucose monitor (Roche, Madison, WI).
Food intake and starvation
Rats were given weighed food containing their 100, 50, or 25% of their normal food requirement (in each diet group 100, 50, and 25) for 10 days followed by 48-h complete fasting. Normal food requirement of rat was calculated as average of daily food intake of 10 normal rats in 10 days.
Data were analyzed using one-way and two-way ANOVA for repeated measure followed by the Bonferroni Post hoc test. Values are Means ± s.e.m. and P values of ≤0.05 were considered statistically significant.
After food restriction, rats fed 50 and 25% ad libitum exhibited a marked reduction in body weight as compared with rats fed ad libitum for 10 days followed by 48-h fasting. (Table 1).
Table 1. Mean body weight before and after diet and percent of body weight reduction in groups which received 100, 50, and 25% of energy requirement for 10 days followed by 48-h fasting. Values are means ± s.e.m.
ACTH, Cor, and Ep concentration after feeding 100, 50, and 25% of their energy needs followed by 48-h fasting have shown in Table 2. ACTH, Cor, and Ep concentration was greater (P ≤ 0.05) in rats fed 50% than in 100 and 25% fed ad libitum. By 48-h fasting, plasma ACTH, Cor, and Ep concentration increased in normal and 50% starved groups, but the percent of increment was less in normal than in 50% starved rats (P ≤ 0.05). Surprisingly, in 25% fed ad libitum we have not seen any significant change in all of this hormones (Table 2).
Table 2. Mean plasma concentration of ACTH, corticosterone, and epinephrine in groups which received 100, 50, and 25% of energy requirement for 10 days followed by 48-h fasting. Values are means ± s.e.m.
Effect of fasting on basal mean plasma concentration of ghrelin, orexin, and galanin
The basal mean plasma concentration of ghrelin, orexin, and galanin was higher in fasted rats (25 and 50% ad libitum) comparing with the normal diet (ad libitum).
Effect of Cor, Ep, and combination of both on ghrelin in different diets
As shown in Figure 1a, circulating ghrelin levels were increased significantly in rats fed 25% ad libitum 30 min after a single injection of Cor (3 µg/kg BW) as compared with other diet groups (P ≤ 0.05). Mean plasma concentration of ghrelin in 50 and 100% food ad libitum rats were significantly reduced 30 min post injection of Ep (3 µg/kg BW) (P ≤ 0.05), although rats fed 25% ad libitum did not show any significant change as compared with preinjection (P ≥ 0.05) (Figure 1b). Combination of Ep and Cor (3 µg/kg BW) decreased the circulating ghrelin levels in rats fed 50 and 100%, 60 min after injection as compared with preinjection (P ≤ 0.05), although they do not affect on ghrelin concentration in 25% starved rats. (Figure 1c).
Effect of Cor, Ep, and combination of both on orexin in different diets
No detectable alterations in circulating orexin concentration were observed in rats received Cor (3 µg/kg BW) in any diet groups (P ≥ 0.05) (Figure 2a). In 50% food-restricted rats, orexin levels were statistically decreased 120 min after Ep injection (3 µg/kg BW) (P ≤ 0.05) and not anymore changed in other diet groups (P ≥ 0.05) (Figure 2b). Orexin levels in rats fed ad libitum decreased significantly 120 min after injection of Cor and Ep (3 µg/kg BW) (P ≤ 0.05), although, they did not show any significant change in other diet groups (P ≥ 0.05) (Figure 2c).
Effect of Cor, Ep, and combination of both on galanin in different diets
Just In 50% food-restricted rats, galanin levels were decreased 120 min after Cor (3 µg/kg BW) injection (P ≤ 0.05) but they did not show any change after Ep (3 µg/kg BW) injection in any diet groups (P ≥ 0.05) (Figure 3a,b). Combination of Cor and Ep (3 µg/kg BW) reduced galanin concentration 120 min after injection in normal and 50% ad libitum rats (P ≤ 0.05) with no change in rats fed 25% (P ≥ 0.05) (Figure 3c).
Effect of Cor, Ep, and combination of both on glucose in different diets
Cor, Ep, and combination of both (3 µg/kg BW) significantly increased the level of blood glucose concentration in rats fed 100 and 50% ad libitum, 120, 60, and 30 min postinjection, respectively (P ≤ 0.05) (Figure 4a,b). But they was no change in blood glucose concentration in 25% ad libitum rats after injections (P ≥ 0.05) (Figure 4c).
Our data are consistent with the hypothesis that ghrelin, orexin, and galanin have a role in long-term regulation of body weight and mean plasma concentration of them were high in diet induced weight loss. As we have shown in this study, basal mean plasma concentration of ghrelin, orexin, and galanin were higher in rats fed 50 and 25% than those fed ad libitum (Table 1). Many studies have shown the role of ghrelin on fasting, meal initiation and long-term weight regulation (1,2). In addition, circulating ghrelin and orexin concentrations are negatively correlated with BMI in humans (3,8). Galanin involves in energy balance controlling by decreasing energy expenditure (4). This finding confirmed the role of ghrelin, orexin, and galanin in long-term body weight and energy balance controlling. However, it is interesting that basal ghrelin, orexin, and galanin in rats fed 25% ad libitum which shows more weight loss is lower than rats with 50%. This finding may suggest that, the relation between them and BMI is not simple adverse.
Previous studies have shown that both circulating forms of ghrelin are sensitive to various stressors. Exposure to acute physical stressors (abdominal surgery, immunological/endotoxin injection, and exercise) reduced, whereas metabolic (cold exposure, fasting, and caloric restriction) and psychological stressors increased ghrelin levels (13), although some study even show no changes, for example exposing male volunteers to the standardized laboratory stressors involving public speaking on 2 days had no effect on ghrelin (14). In this study, using experimental paradigm, we have shown that Ep inhibits ghrelin, orexin, and galanin secretion but we have not seen any effect for Cor. However, although there is no change in ghrelin, orexin, and galanin levels by Cor but it seems by results that Cor has an additive effect on Ep, because ghrelin reduction in rats infused by Ep and Cor appear 60 min vs. 120 min in Ep injection condition. Also in term of galanin, Ep injection did not change it. But administration of Ep and Cor reduced galanin 120 min after injection. However we have not seen additive effect for Cor in orexin secretion. Surprisingly, the reduction of ghrelin, orexin, and galanin neither by Ep nor by Cor have not seen in rats fed 25% ad libitum.
It has been shown that ghrelin, orexin, and galanin reduction caused by Ep injection is associated with severely increased levels of blood glucose. Previous studies have shown that Ep can increase blood glucose concentration (15). On the other hand, oral or i.v. injection of glucose reduces plasma concentration of ghrelin (16). These findings raise the possibility that increment of glucose is one mechanism by which Ep reduces ghrelin. Surprisingly, Cor, Ep, and ACTH concentrations at 48-h fasting in rats fed 25% ad libitum is lower than rats fed normal or 50% ad libitum, although baseline glucose in rats fed 25% is higher than others. On the other hand, injection of Ep, Cor, and combination of both, did not change glucose secretion in this group. It all shows that increment of baseline glucose in this diet group is not dependent to Ep and Cor secretion. High baseline glucose in this group may be the consequence of insulin secretion impairment. However it needs future study to be well understood.
Moreover, insulin is increased by Ep directly in humans (17) or indirectly by glucose increment. Several studies have shown a close relationship between insulin, ghrelin, and galanin secretion. Insulin intracerebroventricular infusion decreased ghrelin concentration in healthy subjects and galanin gene expression in hypothalamus (18,19). Our finding of markedly reduced ghrelin, orexin, and galanin associated with glucose increment suggests that glucose and insulin can now be studied as potential mechanism by which this procedure causes Ep induced ghrelin, orexin, and galanin suppression. Therefore, we can suggest that stress is a mechanism involving in short- and long-term regulation of energy balance.
Several pieces of evidence suggest that one mechanism by which increasing in Ep lead to suppression of ghrelin, orexin, and galanin may be via direct engagement of leptin. For instance, Ep increases leptin transport across the blood-brain barrier (20). On the other hand, leptin has an inhibitory effect on ghrelin, orexin, and galanin. For example, intracerebroventricular injection of leptin reduced orexin in lateral area of hypothalamus (21,22) also, exist of leptin receptors on galanin and neuropeptide Y producing cells suggest that leptin has a direct effect on galanin neurons (23,24). galanin gene expression in hypothalamus was decreased by leptin intracerebroventricular injection which is associated by inhibition of food intake and weight loss (25,26). Hyperleptinemia prevents the increase of plasma ghrelin during moderate short-term caloric restriction (27). Taken together, all these data suggest that leptin may be a target of Ep in the regulation of feeding.
We have identified that diminished circulating levels of ghrelin, orexin, and galanin by Ep is restricted to the metabolic state. Since, rats fed normal and 50% ad libitum showed same response to treatments but rats with 25% ad libitum did not show any response. It is noteworthy that blood glucose did not change in rats fed 25% ad libitum by any treatment too, whereas in other diet groups, treatments caused strongly increased blood glucose concentration. Therefore, here we provide some evidence that very sever diet may disturb normal energy balance regulation systems. It means that mechanism involved in short- and long-term energy balance are only responsible in normal metabolic states. One study showed that reduced caloric intake, weight loss, or catabolic state have powerful effects on several endocrine systems as 3-week severe fasting have shown disturbances in hypothalamic-pituitary-adrenal functions, with elevated plasma Cor level, and insufficient suppression following 1.5 mg dexamethasone. Also during fasting basal thyroid-stimulating hormone values were lowered and the thyroid-stimulating hormone response to thyrotropin-releasing hormone was blunted and the plasma level of growth hormone was elevated (28). Data from present study suggest that energy balance homeostasis mechanisms may affected by metabolic state too.
In summary, we found that stress decreased neurotransmitters involved in food intake, and Ep is the key hormone for this effect although, Cor may have an additive effect. Also this effect may be mediated by blood glucose and insulin, since in groups that blood glucose did not change, the ghrelin, orexin, and galanin did not change too. On the other hand, it seems that glucose secretion in rats fed 25% ad libitum is not stimulated by Ep or Cor. All of this is showing that stress system may act as a food intake controlling mechanism in different condition and any problem in this process, in secretion level or factors involved on functions, can disturb energy balance controlling mechanism. On the other hand, we found that metabolic situation has an affection on energy balance regulating systems function as this ability destroyed in very sever diet.