Involvement of cholinergic neurons in the regulation of the ghrelin secretory response to feeding in sheep

We previously demonstrated that a transient surge in plasma levels of ghrelin occurs just prior to a scheduled meal and that this surge is modified by the feeding regimen. This suggests that the ghrelin secretion is regulated by the autonomic nervous system, especially the cholinergic projections to the stomach. To test this hypothesis, we investigated changes in plasma ghrelin levels at feeding time in rams by administering cholinergic blockers (atropine and hexamethonium) and a cholinergic accelerator (metoclopramide). The average food intake in each group infused with atropine, hexamethonium, metoclopramide, and saline was 150+/-28, 137+/-46, 153+/-50, and 1075+/-25g, respectively. Plasma ghrelin concentrations increased (P<0.05) after i.v. infusion of hexamethonium and gradually decreased (P<0.05) after i.v. infusion of metoclopramide. Plasma ghrelin levels in hexamethonium-treated animals were greater (P<0.05) than those of atropine-treated animals. Plasma ghrelin levels were significantly (P<0.05) higher in sheep given i.v. infusions of atropine or hexamethonium than the levels in normal- or pair-fed sheep infused with saline. Plasma ghrelin levels were similar in metoclopramide-treated, pair-fed, and control animals. These results support the possibility that ghrelin secretion is regulated by cholinergic neurons of the vagus and that cholinergic activity suppresses ghrelin secretion in sheep.     

Enriched protein diet-modified ghrelin expression and secretion in rats

Gastrointestinal (GI) integrity and function are regulated by nutrition and growth factors. The discovery of ghrelin, a natural growth hormone (GH) secretagogue produced by the gastrointestinal (GI) tract, is a potential link between diet and growth signals. The aim of this study was to evaluate macronutrient effect on ghrelin expression and secretion in addition to some possible function in intestinal trophic status. Wistar rats were fed a high-carbohydrate, high-protein (HP), high-fat or standard (St) diet. Animals received the same daily food volume and caloric intake. After 7 days, animals were fasted for 24 h and blood and tissue samples were obtained just before feeding or at 2 or 6 h after feeding. Fasting high-protein-fed rats had higher ghrelin plasma levels than with rats fed the high-carbohydrate, high-fat or standard diets. Two-hours after refeeding, ghrelin plasma levels had decreased in all groups with a slight recovery at 6 h after refeeding, except in the high-protein group. Ghrelin plasma levels in rats fed with the high-protein diet correlated negatively with their GH and insulin-like growth factor 1 (IGF-1) plasma concentrations which were also the lowest among the study groups. In conclusion, ghrelin secretion was nutritionally manipulated because a protein-enriched diet increased its levels.     

Circulating leptin response to feeding and exogenous infusion of insulin in sheep exposed to thermoneutral and cold environments

Leptin has been shown to regulate feed intake and energy expenditure. Insulin stimulates leptin secretion in rodents, but its action on leptin secretion is still obscure in ruminants. If insulin stimulates leptin secretion in ruminants, circulating leptin concentrations may change during exposure to cold, because of fluctuating insulin secretion and action in the cold environment. The present experiment was designed to determine whether feeding or exogenous administration of insulin affects circulating leptin levels in sheep exposed to thermoneutral and cold environments. Suffolk rams that were shorn and fed a diet once daily were subjected to a thermoneutral (20 degrees C) or cold (0 degrees C) environment for at least 1 week. Overall mean concentrations of plasma leptin in the feeding experiment were lower (P<0.05) in the cold environment than in the thermoneutral environment. Plasma leptin levels remained relatively unchanged after feeding in both environments, though plasma insulin response to feeding in both environments increased (P<0.01). The euglycemic clamps (insulin infusion rate: 4 mUkgBW(-1)min(-1) for 2 h) increased (P<0.01) circulating leptin concentrations in the thermoneutral, but not in the cold environment. These results suggest that lower circulating leptin levels in ruminants exposed to the cold environment could be partly due to the depressed insulin action on leptin secretion.     

Effects of dietary zinc deficiency on gonadotrophin secretion and testicular growth in young male sheep.

The hypothesis that the secretion of gonadotrophins would be reduced by zinc deficiency was tested in five groups of four young Merino rams (initial liveweight 22 kg). Four groups were fed ad libitum with diets containing 4, 10, 17 or 27 micrograms Zn g-1. The effects of loss of appetite on the deficient diet was controlled by feeding a fifth group (pair-fed control) at a rate of 27 micrograms Zn g-1, but the amount of feed offered was restricted to that eaten voluntarily by the deficient (4 micrograms Zn g-1) group. Blood was sampled every 20 min for 32 h on two occasions before the treatments were imposed and 96 days later, at the end of the experiment. The rams were injected with gonadotrophin-releasing hormone (GnRH; 10 ng kg-1 i.v.) after each serial sampling, and with naloxone (1 mg kg-1 i.v.) 24 h after the end of the final GnRH test. In the group that were fed the diet with the lowest zinc content, the concentration of zinc in blood plasma was reduced to 18% of that in the pair-fed controls (P < 0.05) and was within the deficient range. The appetite of the deficient rams was half that of the controls fed 27 micrograms Zn g-1 ad libitum and there was no increase in liveweight or testicular diameter during pubertal development. Similar, but smaller, effects were observed in the pair-fed controls. There were no significant differences between pair-fed and deficient groups in the frequency of the luteinizing hormone (LH) pulses or in the concentration of follicle-stimulating hormone (FSH), but the secretion of gonadotrophins was markedly lower in both groups than in the control rams fed ad libitum. The response to GnRH was not affected by treatment, but the increase in LH pulse frequency evoked by naloxone was lower in the deficient animals than in other groups. The animals fed zinc at intermediate rates (10-17 micrograms g-1) showed similar responses to the controls fed ad libitum. It is concluded that the specific effects of zinc deficiency on testicular function were small. Most of the reduction in testicular growth in rams fed a deficient diet was not specifically related to the trace element, but was due to the fall in energy and protein intake caused by the loss of appetite. This leads to a reduction in the frequency of GnRH pulses secreted by the hypothalamus, and to low rates of gonadotrophin secretion by the pituitary gland.     

Effects of level of feeding and progesterone dose on plasma and faecal progesterone in ovariectomised cows

The effects of two levels of feeding and two doses of progesterone (P4) on plasma and faecal progesterone metabolites (FP4M) were studied using a total of 24 ovariectomised (OVX), non-lactating, Holstein-Friesian cows. Cows were grazed on improved ryegrass/white clover pastures and allowed ad libitum access to pasture or were restricted to grazing for a total of 4 h per day in two 2 h periods. Progesterone (P4) was administered as one or two, simultaneous, intravaginal progesterone devices (CIDR). The cows were adapted to their pasture supply for 2 weeks before the start of the progesterone treatments. The progesterone devices were administered for 11 days and the cows were dosed with slow release chromic oxide capsules during the P4 treatment to allow faecal output (FO) to be estimated. Daily blood samples for P4 assay and weekly samples for blood metabolite assay were collected. Faecal samples were collected per rectum daily and assayed for pregnanes containing a 20-oxo-, 20alpha- or a 20beta-OH group by enzyme immunoassay (EIA). Daily FO was higher (P < 0.001) for ad libitum than pasture restricted cows (6.3 vs 4.1 kg DM) but was similar for both doses of P4. The average mass of P4 released from a CIDR device over a 11-day period was higher for cows allowed ad libitum pasture compared with those on restricted pasture (0.64 vs 0.60 g; P = 0.04). Plasma P4 concentrations, however, were higher in restricted than ad libitum fed cows (1x CIDR: 1.81 vs 1.41 ng/ml; 2x CIDR: 4.10 vs 3.46 ng/ml). Increasing the progesterone dose significantly (P < 0.001) increased both the concentrations and daily totals of the faecal pregnanes assayed and total FP4M. Restricted pasture cows had higher (P < 0.001) pregnanes and FP4M concentrations than cows fed ad libitum. Daily total faecal pregnane and FP4M did not differ between feeding levels except for faecal 20alpha-pregnane which was highest for ad libitum fed cows (P < 0.05). These results showed that the plasma concentrations of P4 in CIDR-treated OVX cows were negatively associated with the level of feeding. Level of feeding and dose of P4 affected the concentrations of FP4M, but the daily excretion rate of FP4M was not positively influenced by the level of feeding.     

Effects of feeding state on glycaemic and insulinaemic responses to a starchy meal in horses: a methodological approach

A standardised methodology is required for classification of starchy diets. One important question is what feeding status is optimal to describe glycaemic and insulinaemic responses to the respective starchy diet. Four horses were fed, in a randomised order, four different feeding protocols relative to offering hay before or after cracked corn (CC): (i) ad libitum hay for 12 h before feeding CC and ad libitum hay after CC intake for 9 h (+CC+), (ii) ad libitum hay for 12 h before feeding CC and no hay after CC intake for 9 h (+CC-), (iii) feed restriction for 12 h before feeding CC and ad libitum hay after CC intake for 9 h (-CC+) and (iv) 1.2 kg hay/100 kg body weight (BW) per day, divided into two equal portions and offered at 0900 h and 1900 h, feed restriction for 12 h before feeding CC and no hay after CC intake for 9 h (-CC-). CC intake was adjusted to a starch intake of 2 g/kg BW. The different hay offerings did not affect basal plasma glucose and insulin levels. A significant rise in plasma glucose and insulin was found after CC intake for all diets. The highest peak glucose levels were analysed for -CC+, and the lowest glucose peaks were found for +CC- (diet P < 0.05). The highest insulin peaks were monitored for -CC+ (31.27 ± 18.19 μU/ml) and lower peaks for +CC- (13.36 ± 2.93 μU/ml) (diet P < 0.05). Insulin for -CC- and +CC- returned to resting values about 300 min after CC feeding. For +CC+ and -CC+, insulin levels were still above resting levels 510 min after CC intake (diet P < 0.05). The present data suggest that feed restriction for 12 h before feeding the starchy diet and no further roughage intake during blood sampling period provide the best-defined conditions.     

Effects of chronic food restriction and treatments with leptin or ghrelin on different reproductive parameters of male rats

The existence of a close relationship between energy status and reproductive function is well-documented, especially in females, but its underlying mechanisms remain to be fully unfolded. This study aimed to examine the effects of restriction of daily calorie intake, as well as chronic treatments with the metabolic hormones leptin and ghrelin, on the secretion of different reproductive hormones, namely pituitary gonadotropins and prolactin, as well as testosterone, in male rats. Restriction (50%) in daily food intake for 20 days significantly reduced body weight as well as plasma PRL and T levels, without affecting basal LH and FSH concentrations and testicular weight. Chronic administration of leptin to rats fed ad libitum increased plasma PRL levels and decreased circulating T, while it did not alter other hormonal parameters under analysis. In contrast, in rats subjected to 50% calorie restriction, leptin administration increased plasma T levels and reduced testis weight. Conversely, ghrelin failed to induce major hormonal changes but tended to increase testicular weight in fed animals, while repeated ghrelin injections in food-restricted males dramatically decreased plasma LH and T concentrations and reduced testis weight. In sum, we document herein the isolated and combined effects of metabolic stress (50% food restriction) and leptin or ghrelin treatments on several reproductive hormones in adult male rats. Overall, our results further stress the impact and complex way of action of different metabolic cues, such as energy status and key hormones, in reproductive function also in the male.     

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats

Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48h fasted rats and 48h fasted rats refed 2h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.     

Melatonin and corticosterone regulation: Feeding time or the light:Dark cycle?

Under ad libitum feeding, male rats exhibit a marked rhythm of plasma and pineal melatonin; levels are low during the day and high at night. Restricting food availability to a 2 hour period during the light or dark does not markedly influence the melatonin rhythm, both groups having a crest in circulating melatonin during the dark. In contrast, plasma corticosterone levels are influenced by both the light-dark cycle and feeding. Animals fed early in the light period exhibit a biomodal corticosterone secretory pattern, with high steroid levels immediately prior to feeding and again just before lights-out, animals fed early in the dark have a single crest, just before food presentation. These data provide evidence for the dissociation of melatonin and corticosterone secretory patterns, providing support for the hypothesis that multiple regulators control neuroendocrine rhythmicity.     

Time-restricted feeding entrains daily rhythms of energy metabolism in mice

Energy metabolism, oxygen consumption rate (VO2), and respiratory quotient (RQ) in mice were monitored continuously throughout 12:12-h light-dark cycles before, during, and after time-restricted feeding (RF). Mice fed ad libitum showed robust daily rhythms in both parameters: high during the dark phase and low during the light phase. The daily profile of energy metabolism in mice under daytime-only feeding was reversed at the beginning of the first fasting night. A few days after daytime-only feeding began, RF also reversed the circadian core body temperature rhythm. Moreover, RF for 6 consecutive days shifted the phases of circadian expression patterns of clock genes in liver significantly by 8-10 h. When mice were fed a high-fat (HF) diet ad libitum, the daily rhythm of RQ dampened day by day and disappeared on the sixth day of RF, whereas VO2 showed a robust daily rhythm. Mice fed HF only in the daytime had reversed VO2 and RQ rhythms. Similarly, mice fed HF only in the daytime significantly phase shifted the clock gene expression in liver, whereas ad libitum feeding with HF had no significant effect on the expression phases of liver clock genes. These results suggested that VO2 is a sensitive indicator of entrainment in the mouse liver. Moreover, physiologically, it can be determined without any surgery or constraint. On the basis of these results, we hypothesize that a change in the daily VO2 rhythm, independent of the energy source, might drive phase shifts of circadian oscillators in peripheral tissues, at least in the liver.     

Chronic ethanol feeding inhibits plasma levels of insulin-like growth factor-1

The purpose of this study was to determine whether the generalized catabolic effects of chronic ethanol may be associated with a decline in plasma levels of insulin-like growth factor-1 (IGF-1). Male Sprague-Dawley rats were fed a liquid diet containing 5% ethanol or pair-fed a diet made isocaloric with maltose-dextrin. Animals were maintained on this diet for either 12 days or 4.5 months. Another group of animals were fed control diet ad libitum for 2 weeks. After 12 days of feeding, plasma concentrations of IGF-1 in ad libitum fed rats were 771 +/- 41 ng/ml which was greater than concentrations in either pair-fed (595 +/- 23 ng/ml) or ethanol-fed (680 +/- 40 ng/ml) rats (P less than 0.05). After 4.5 months of feeding, plasma levels of IGF-1 in ad libitum and pair-fed rats were similar to the 12 day study (736 +/- 56 and 607 +/- 26 ng/ml, respectively). However, a significant decrease in plasma levels of IGF-1 was observed in ethanol-fed animals over the 4.5 month period (551 +/- 28 ng/ml, P less than 0.05). Results of a similar study in rats fed a high-fat diet for 4.5 months were similar to those found with the low-fat diet. These results indicate that 1) dietary restriction of the type routinely used in this pair-feeding regimen decreases plasma levels of IGF-1, 2) chronic ethanol feeding further decreases plasma IGF-1 levels compared to pair-fed rats, 3) the effects of ethanol on IGF-1 concentrations are not modified by dietary fat, and 4) the effects on IGF-1 are not directly dependent on elevated plasma ethanol concentrations. Our results suggest that IGF-1 secreting cells in the liver may be progressively damaged by chronic ethanol feeding.     

Influence of rest and exercise at a simulated altitude of 4,000 m on appetite, energy intake, and plasma concentrations of acylated ghrelin and peptide YY

The reason for high altitude anorexia is unclear but could involve alterations in the appetite hormones ghrelin and peptide YY (PYY). This study examined the effect of resting and exercising in hypoxia (12.7% O(2); ～4,000 m) on appetite, energy intake, and plasma concentrations of acylated ghrelin and PYY. Ten healthy males completed four, 7-h trials in an environmental chamber in a random order. The four trials were control-normoxia, control-hypoxia, exercise-normoxia, and exercise-hypoxia. During exercise trials, participants ran for 60 min at 70% of altitude-specific maximal oxygen consumption (Vo(2max)) and then rested. Participants rested throughout control trials. A standardized meal was consumed at 2 h and an ad libitum buffet meal at 5.5 h. Area under the curve values for hunger (assessed using visual analog scales) tended to be lower during hypoxic trials than normoxic trials (repeated-measures ANOVA, P = 0.07). Ad libitum energy intake was lower (P = 0.001) in hypoxia (5,291 ± 2,189 kJ) than normoxia (7,718 ± 2,356 kJ; means ± SD). Mean plasma acylated ghrelin concentrations were lower in hypoxia than normoxia (82 ± 66 vs. 100 ± 69 pg/ml; P = 0.005) while PYY concentrations tended to be higher in normoxia (32 ± 4 vs. 30 ± 3 pmol/l; P = 0.059). Exercise suppressed hunger and acylated ghrelin and increased PYY but did not influence ad libitum energy intake. These findings confirm that hypoxia suppresses hunger and food intake. Further research is required to determine if decreased concentrations of acylated ghrelin orchestrate this suppression.     

The effect of a pharmaceutical ghrelin agonist on lifespan in C57BL/6J male mice: A controlled experiment

Interventions for animal lifespan extension like caloric restriction (CR) have identified physiologic and biochemical pathways related to hunger and energy-sensing status as possible contributors, but mechanisms have not been fully elucidated. Prior studies using ghrelin agonists show greater food intake but no effect on lifespan in rodent models. This experiment in male C57BL/6J mice tested the influence of ghrelin agonism for perceived hunger, in the absence of CR, on longevity. Mice aged 4 weeks were allowed to acclimate for 2 weeks prior to being assigned (N = 60/group). Prior to lights off daily (12:12 cycle), animals were fed a ghrelin agonist pill (LY444711; Eli Lilly) or a placebo control (Ctrl) until death. Treatment (GhrAg) animals were pair-fed daily based on the group mean food intake consumed by Ctrl (ad libitum feeding) the prior week. Results indicate an increased lifespan effect (log-rank p = 0.0032) for GhrAg versus placebo Ctrl, which weighed significantly more than GhrAg (adjusted for baseline weight). Further studies are needed to determine the full scope of effects of this ghrelin agonist, either directly via increased ghrelin receptor signaling or indirectly via other hypothalamic, systemic, or tissue-specific mechanisms.     

Changes in the jugular haematocrit of sheep during feeding.

Intact and splenectomized sheep with and without a rumen fistula were used to investigate changes in the jugular blood haematocrit and plasma osmolality during hourly and once-daily feeding regimes. Osmolality was also estimated in the ruminal fluid of fistulated sheep with spleens. Haematocrit decreased in sheep with spleens before they were given a once-daily feed; it increased when these sheep started to feed, reaching a maximum increase of 13% after 30 min of feeding; it decreased during the remaining 45 min of feeding time and usually continued to decrease after feeding stopped. These changes were not due to diurnal influences. Splenectomized sheep fed once daily showed only small decreases in haematocrit before they were fed. Increases occurred with the onset of eating but they were smaller (7%) than in intact sheep and were of shorter duration. In hourly fed sheep with spleens, haematocrit decreased in the early stages of sampling in a manner similar to that for sheep fed once daily. The changes in haematocrit that did occur were not related in any obvious manner to the feeding regime. The haematocrit in splenectomized sheep fed hourly was stable throughout feeding. Variations in the haematocrit in splenectomized sheep, equivalent to a range of 13% in one of them, were observed in a series of blood samples obtained during a 5-h period remote from the feeding time. Large increases occurred in osmolality of ruminal fluid when sheep were fed daily and this was abolished by hourly feeding. Plasma osmolality in sheep fed once daily increased slowly. Maxima occurred after 100 min from the start of eating and were 7% greater than prefeeding values. Only minor changes were observed when these sheep were fed hourly.     

Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems

The activities of rat intestinal enzymes, sucrase, lactase, maltase, trehalase, γ-glutamyltransferase, leucylnaphthylamide-hydrolyzing activity, and the transport system for glucose follow diurnal rhythms on ad libitum and restricted feeding regimes. In response to 6 days of restricted feeding, food available between 1400 and 1800 Eastern Standard Time, all rhythms shifted in time and the daily levels of activities were changed. Alkaline phosphatase activity followed a diurnal rhythm only in restricted fed animals.In restricted fed rats several activity patterns were observed, some with short periods of maximum activity, 3 h or less, and some with plateaus of maximum activity, 5–9 h long. In respect to the time of day of the synchronizer, sucrase peaked before feeding, glucose transport peaked during feeding, alkaline phosphatase peaked after feeding, and the other enzymes had higher levels of activity before, during and after feeding. The effect of restricted feeding on the daily activity levels were: a decrease in leucylnaphthylamide-hydrolyzing activity, no change in alkaline phosphatase, and increases in the others.These enzyme and transport systems exhibit a large amount of individual regulation or control as reflected by the lack of a uniform activity pattern and response to the synchronizer, and the variation in direction and magnitude of the adaptations to restricted feeding.     

Effects of moderate hyperthyroidism and time relative to feeding on tissue responsiveness to insulin in sheep

The effects of moderate hyperthyroidism and time relative to feeding on tissue responsiveness to insulin were determined in four sheep using a hyperinsulinemic euglycemic clamp procedure, because thyroid hormones have glucoregulatory function in ruminants and non-ruminant animals. The sheep were fed alfalfa hay cubes and corn-based concentrates. They were intravenously injected in turn with saline (control treatment) and then with triiodothyronine (1.5 nmol d(-1); T3 treatment) once daily for 23 days for each treatment. The glucose clamp procedure was performed at four different times relative to feeding (before, during and after feeding) in both treatments. Insulin was intravenously infused at a constant rate of 6.0 mU (kgxmin)(-1) for 2 h and a glucose solution was infused to maintain euglycemia. Plasma T3 concentrations were higher (P=0.0001) for the T3 treatment than for the control treatment (4.0 and 1.3 nmol l(-1), respectively). For the glucose clamp procedure, glucose infusion rates were greater (P=0.0001) for the T3 treatment than for the control treatment, and were greater (P<0.05) during feeding than before feeding and after the end of feeding, but no interaction was detected. In conclusion, tissue responsiveness to insulin in sheep was independently enhanced by moderate hyperthyroidism induced by intravenous T3 injection and feeding.     

Scheduled feeding results in adipogenesis and increased acylated ghrelin

Ghrelin, known to stimulate adipogenesis, displays an endogenous secretory rhythmicity closely related to meal patterns. Therefore, a chronic imposed feeding schedule might induce modified ghrelin levels and consequently adiposity. Growing Wistar rats were schedule-fed by imposing a particular fixed feeding schedule of 3 meals/day without caloric restriction compared with total daily control intake. After 14 days, their body composition was measured by DEXA and compared with ad libitum-fed controls and to rats daily intraperitoneal injection with ghrelin. Feeding patterns, circadian activity, and pulsatile acylated ghrelin variations were monitored. After 14 days, rats on the imposed feeding schedule displayed, despite an equal daily calorie intake, a slower growth rate compared with ad libitum-fed controls. Moreover, schedule-fed rats exhibiting a feeding pattern with intermittent fasting periods had a higher fat/lean ratio compared with ad libitum-fed controls. Interestingly, ghrelin-treated rats also showed an increase in fat mass, but the fat/lean ratio was not significantly increased compared with controls. In the schedule-fed rats, spontaneous activity and acylated ghrelin levels were increased and associated with the scheduled meals, indicating anticipatory effects. Our results suggest that scheduled feeding, associated with intermittent fasting periods, even without nutrient/calorie restriction on a daily basis, results in adipogenesis. This repartitioning effect is associated with increased endogenous acylated ghrelin levels. This schedule-fed model points out the delicate role of meal frequency in adipogenesis and provides an investigative tool to clarify any effects of endogenous ghrelin without the need for ghrelin administration.     

Fasting ghrelin does not predict food intake after short-term energy restriction

Objective: To study the role of ghrelin as a hunger signal during energy restriction and to test the hypothesis that changes in fasting leptin concentrations during energy restriction are associated with changes in fasting ghrelin concentrations. Research Methods and Procedures: Thirty‐five healthy, lean men (23 ± 3 years of age; BMI: 22.3 ± 1.6 kg/m²) participated in a controlled intervention study. Fasting ghrelin and leptin concentrations were measured before and after 2 days of 62% energy restriction and after a 2‐day period of ad libitum food intake. Energy intake during the latter period was assessed. Results: On average, ghrelin concentrations did not change (0.05 μg/liter; 95% confidence interval, ?0.03; 0.12) during energy restriction. Changes in ghrelin concentration during energy restriction were not associated with energy intake during the ad libitum period (r = 0.07; not significant). Ad libitum energy intake was, however, associated with the change in ghrelin concentrations during the same period (r = ?0.34; p = 0.05). Ghrelin and leptin concentrations were not associated. In addition, the ratio of percentage changes in ghrelin and leptin during energy restriction was not correlated with ad libitum food intake after energy restriction (r = ?0.26; p = 0.14). Discussion: Fasting ghrelin concentrations did not rise after a 2‐day energy restriction regimen. Moreover, changes in ghrelin concentrations during energy restriction were not associated with subsequent ad libitum food intake, suggesting that fasting ghrelin does not act as a hunger signal to the brain. The data did not support our hypothesis that leptin suppresses ghrelin levels.     

Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems.

The activities of rat intestinal enzymes, sucrase, lactase, maltase, trehalase, gamma-glutamyltransferase, leucylnaphthylamide-hydrolyzing activity, and the transport system for glucose follow diurnal rhythms on ad libitum and restricted feeding regimes. In response to 6 days of restricted feeding, food available between 1400 and 1800 Eastern Standard Time, all rhythms shifted in time and the daily levels of activities were changed. Alkaline phosphatase activity followed a diurnal rhythm only in restricted fed animals. In restricted fed rats several activity patterns were observed, some with short periods of maximum activity, 3 h or less, and some with plateaus of maximum activity, 5-9 h long. In respect to the time of day of the synchronizer, sucrase peaked before feeding, glucose transport peaked during feeding, alkaline phosphatase peaked after feeding, and the other enzymes had higher levels of activity before, during and after feeding. The effect of restricted feeding on the daily activity levels were: a decrease in leucylnaphthylamide-hydrolyzing activity, no change in alkaline phosphatase, and increases in the others. These enzyme and transport systems exhibit a large amount of individual regulation or control as reflected by the lack of a uniform activity pattern and response to the synchronizer, and the variation in direction and magnitude of the adaptations to restricted feeding.     

Plasma ghrelin levels and hunger scores in humans initiating meals voluntarily without time- and food-related cues

Ghrelin is an orexigenic hormone that is implicated in meal initiation, in part because circulating levels rise before meals. Because previous human studies have examined subjects fed on known schedules, the observed preprandial ghrelin increases could have been a secondary consequence of meal anticipation. A causal role for ghrelin in meal initiation would be better supported if preprandial increases occurred before spontaneously initiated meals not prompted by external cues. We measured plasma ghrelin levels among human subjects initiating meals voluntarily without cues related to time or food. Samples were drawn every 5 min between a scheduled lunch and a freely requested dinner, and hunger scores were obtained using visual analog scales. Insulin, glucose, fatty acids, leptin, and triglycerides were also measured. Ghrelin levels decreased shortly after the first meal in all subjects. A subsequent preprandial increase occurred over a wide range of intermeal intervals (IMI; 320-425 min) in all but one subject. Hunger scores and ghrelin levels showed similar temporal profiles and similar relative differences in magnitude between lunch and dinner. One subject displayed no preprandial ghrelin increase and was also the only individual whose insulin levels did not return to baseline between meals. This finding, along with a correlation between area-under-the-curve values of ghrelin and insulin, suggests a role for insulin in ghrelin regulation. The preprandial increase of ghrelin levels that we observed among humans initiating meals voluntarily, without time- or food-related cues, and the overlap between these levels and hunger scores are consistent with a role for ghrelin in meal initiation.