Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

Biostable insect kinin analogs reduce blood meal and disrupt ecdysis in the blood-gorging Chagas’ disease vector,Rhodnius prolixus

Rhodnius prolixus is a blood-gorging hemipteran that takes blood meals that are approximately 10 times its body weight. This blood meal is crucial for growth and development and is needed to ensure a successful molt into the next instar. Kinins are a multifunctional family of neuropeptides which have been shown to play a role in the control of feeding in a variety of insects. In this study, two biostable Aib-containing kinin analogs were tested to see if they interfere with blood-feeding and subsequent development into the next instar. One of the analogs, 1729 (Ac-R[Aib]FF[Aib]WGa), had no effect on the size of the blood meal or on the subsequent molting of the insect into the next instar. This analog also did not interfere with either short-term or long-term diuresis. The second analog, 1728 ([Aib]FF[Aib]WGa), appeared to be an antifeedant. Insects feeding on blood containing this analog (15 μM) only consumed 60% of the blood meal taken by insects fed on blood without analog. Insects feeding on blood containing 1728 had a slower rate of rapid diuresis (diuresis in the first 3–5 h after feeding) leading to less urine being excreted by 5 days post feeding. The consequence of these effects was that insects fed on 1728 did not molt. This data indicates that the biostable Aib-containing analog 1728 disrupts normal growth and development in the blood-feeding insect, R. prolixus.     

Diuretic hormone in adult Rhodnius prolixus: total store and speed of release

Adult Rhodnius take smaller blood meals than do fifth stage larvae and excrete less fluid afterwards. The total amount of diuretic hormone and active precursors that can be extracted from their nervous system is also smaller. However, more diuretic hormone is released in adult than in fifth stage insects when the hormone release sites are depolarized for short periods with potassium-rich solutions. This shows that the adult can release diuretic hormone at a higher rate than can the fifth stage larva, a feature that can be correlated with the larger volume of haemo-lymph which the adult insect has to load with hormone in order to promote diuresis. In vivo , the concentration of diuretic hormone in the haemo-lymph of adult and fifth stage insects rises at the same rate. Detectable amounts of hormone are found in samples of haemolymph from insects which have been feeding for periods as short as 15 s.     

Natriuresis and diuretic hormone synergism in R. prolixus upper Malpighian tubules is inhibited by the anti-diuretic hormone, RhoprCAPA-α2

Insects contain an array of hormones that coordinate the actions of the excretory system to achieve osmotic and ionic balance. In the hematophagous insect, Rhodnius prolixus, two diuretic hormones have been identified, serotonin (5HT) and a corticotropin releasing factor-related peptide (RhoprDH), and both lead to an increase in fluid secretion by Malpighian tubules (MTs). However, only 5HT activates reabsorption by the lower MTs to recover K(+) and Cl(-). An anti-diuretic hormone (RhoprCAPA-α2) is believed to coordinate the cessation of the rapid diuresis following blood meal engorgement. However, the role of RhoprCAPA-α2 on fluid secretion by MTs stimulated by RhoprDH was previously unknown. Here we demonstrate that, unlike the inhibitory effect on 5HT-stimulated secretion by MTs, RhoprCAPA-α2 does not inhibit secretion stimulated by RhoprDH although it does abolish the synergism that occurs between the two diuretic hormones. In addition, we show that the natriuresis elicited by either diuretic hormone is reduced by RhoprCAPA-α2. Using electrophysiological tools, we investigate the possible mechanism by which this complex regulatory pathway is achieved. Analysis of the pH of secreted fluid as well as the triphasic response in transepithelial potential in MTs treated with diuretic hormones, suggests that RhoprCAPA-α2 does not inhibit the V-type H(+) ATPase. Taken together, these results indicate that RhoprCAPA-α2 functions to reduce the rapid diuresis following blood feeding, and in addition, it inhibits the natriuresis associated with diuretic hormone stimulated MTs. This may reflect an important regulatory mechanism related to the slow diuresis that occurs as the K(+)-rich blood cells are digested.     

Can one unrestricted meal buffer the effects of previous pre-meal intervals on the feeding behaviour of sheep?

Varying the time since the last meal is one means of manipulating feeding motivation. In order to use this method effectively it is necessary to know whether and the extent to which effects of one pre-meal interval are carried over to affect the behaviour during the following meals. Pre-meal interval (PMI) is defined here for practical purposes, for short meals, as the time between the start of two successive meals. The possibility that one unrestricted meal might buffer the effects of an 8h as opposed to a 4h PMI on aspects of feeding behaviour was studied with eight Scottish Blackface sheep. They were fed on a regime in which they were given access to food until they finished their meal and lay down (this always occurred within 60min) at which time the remaining food was withdrawn. Feeding behaviour was recorded during the meal after these 4 and 8h intervals, as well as during the following meal 4h later.At a meal after a PMI of 8h, compared to 4h, sheep had a higher intake per meal (mean+/-S.T.D. for 8 and 4h PMI, respectively: 604.4+/-78.8 and 430.1+/-100.9g; P<0.001), a longer meal duration (27.1+/-7.5 and 21.8+/-8.1min; P<0.001), and a tendency for a higher intake rate (23.8+/-6.2 and 21.9+/-8.2g/min; P=0.11). During the following meal 4h later these differences were smaller, but intake per meal still tended to be higher (430.8+/-81.5 and 338.5+/-45.6g; P<0.06) for sheep who had previously had the 8h PMI. Meal duration (21.9+/-7.2 and 20.6+/-7.08min; P=0.28) and intake rate (21.2+/-6.1 and 18.7+/-7.2g/min; P=0.13) were no longer different.A single meal after the different PMIs reduced differences in all three aspects of feeding behaviour observed during the subsequent meal, 4h later, but differences in intake per meal were still apparent. It is suggested that an additional meal may overcome the carry-over effect.     

Developmental changes in Malpighian tubule fluid transport

Developmental changes in Malpighian tubule fluid transport were studied using in vitro and in situ preparations from timed larval, pupal and adult skipper butterflies (Calpodes ethlius). The ability to transport fluid and the rate of fluid secretion depend on the developmental stage and the physiological state. Larval tubules are permanently switched on, do not require a diuretic hormone and transport fluid at an increasing rate as the larvae feed and grow. Fluid transport continues at larval-larval moults but is switched off 24 hr before pupal ecdysis. No secretory activity occurs during the first half of the pupal stage when the tubules are remodelled for adult function. The resumption of fluid transport midway through the pupal stage is in preparation for a rapid diuresis at adult emergence. High rates of fluid secretion are associated with feeding or drinking in adults.     

Temporal patterning of feeding by hummingbirds

For five species of hummingbirds in the laboratory, time between meals was related to energy intake on the first meal and rate of energy expenditure between meals. Field observations gave similar results. Average meal sizes were similar at one intake rate independent of food caloric density; females averaged longer bouts than males. When rate of intake was approximately halved, meal duration approximately doubled and volume intake remained similar. We postulate that feeding is initiated when crop contents reach a lower threshold and that feeding is terminated after ingestion of an optimal volume determined by the added weight of the meal.     

Distention-mediated egg maturation in the mosquito,Aedes aegypti

Abdominal distention accelerates the release of a factor from the head of blood-fed Aedes aegypti mosquitoes. The critical period during which the head is required for oögenesis following blood ingestion is approx 6 h with a 5 μl meal, but small blood meals of 1 μl require the head to be present for significantly longer. Increasing the abdominal distention by supplementing the 1 μl meal with saline results in a critical period similar to that with 5 μl of blood. The information from the distended abdomen appears to travel via the ventral nerve cord. Transection of the ventral nerve cord prevents oögenesis from occurring after small blood meals, but not with larger blood volumes. Topical application of 100 pg of juvenile hormone III can substitute for the distention message.     

The regulation of post-eclosion and post-feeding diuresis in the Monarch butterfly, Danaus plexippus

Both post-eclosion and post-feeding diuresis can be demonstrated in adult Monarch butterflies; and both these processes are significantly inhibited by neck-ligature. The post-eclosion response is restored in neck-ligatured animals by injection of whole head, brain, ventral nerve cord, and corpora cardiaca-corpora allata extracts, and the effect is dose dependent. The active substance appears to be a water soluble, heat stable, trypsin and protease sensitive, polypeptide, with a molecular weight estimated from gel filtration of approx. 3000, that is localized principally in the brain. The amount of active substance present in the head decreases during post-eclosion diuresis, when activity seems to be present in the haemolymph. However, diuretic activity can be demonstrated from heads obtained from Monarchs that are several days or weeks old. In the Monarch, post-eclosion diuresis appears to be under hormonal regulation. By contrast, extracts of whole heads and/or known endocrine organs do not significantly alter post-feeding diuresis in intact or neck-ligatured Monarchs. In addition, although diuresis in response to injections of large volumes of insect saline can be demonstrated in Monarchs, extracts of known endocrine organs do not affect the rate of post-injection diuresis in either neck-ligatured or intact animals. Such experiments, and others involving surgical interruption of the ventral nerve cord, indicate that the eclosion diuretic hormone does not play a major role in the regulation of post-feeding diuresis in this species.     

The structure of feeding behavior in a phytophagous insect (Hylobius abietis)

Analysis of the feeding behavior of animals using such a high temporal resolution that meals can be defined may improve our understanding of the mechanisms regulating feeding. Meals can be distinguished in an ethologically meaningful manner by using the ‘meal criterion’, the shortest non‐feeding interval between feeding bouts recognized as meals. However, such a criterion has only been determined for a few insect species. Applying a recent method developed for assessing meal criteria for vertebrates, we determined the meal criterion for Hylobius abietis (L.) (Coleoptera: Curculionidae) based on data from video recordings of single individuals feeding on seedlings of Norway spruce, Picea abies (L.) Karst. (Pinaceae). The pine weevil is an economically important pest insect, because it feeds on the stem bark of planted conifer seedlings. Weevils had 4–5 meals per day. Each meal lasted about 24 min during which about 13 mm² of bark per meal were removed. Females had longer total meal durations and longer non‐feeding intervals within meals than males. Girdling seedlings did not affect the weevils' feeding properties. The size of meals was significantly correlated with the duration of non‐feeding intervals before and after them. This study is one of few describing the feeding behavior of an insect at a temporal resolution that allows individual meals to be distinguished. With more meal‐related data from insects available, differences in meal properties may be interpreted based on phylogeny, ecology, and physiology. Our results may also assist in the setup and interpretation of studies of plant‐insect interactions, and facilitate the evaluation and development of methods to protect plants against herbivores.     

Effect of solid meal on gastric emptying of,and glycemic and cardiovascular responses to,liquid glucose in older subjects

Gastric emptying is a determinant of the postprandial glycemic and cardiovascular responses to oral carbohydrate. We evaluated the effects of a solid meal on gastric emptying and the glycemic and cardiovascular responses to oral glucose in healthy older subjects. Ten subjects aged 72.1 +/- 1.9 yr were studied. Each subject had measurements of gastric emptying, blood glucose, serum insulin, blood pressure, and heart rate after ingestion of a 50-g glucose drink (300 ml) with (mixed meal) or without (liquid only) a solid meal (300 g ground beef). Gastric emptying of liquid was initially slightly more rapid (P < 0.05) after the mixed meal compared with liquid only at 5 min (92.0 +/- 1.5 vs. 96.0 +/- 1.3%) and much slower (P < 0.05) after 120 min. The time to peak blood glucose was less (39.0 +/- 4.0 vs. 67.5 +/- 10.3 min; P < 0.01) and blood glucose subsequently lower (P < 0.01) after the mixed meal. The increase in serum insulin was greater (P < 0.001) after the mixed meal. Blood pressure fell (P < 0.05) in the first 30 min, with no difference between the two meals. Increase in heart rate after both meals (P < 0.005), was greater (P < 0.05) after the mixed meal. The presence of a noncarbohydrate solid meal had discrepant effects on early and subsequent emptying of a nutrient liquid, which affects postprandial glycemia and increased heart rate.     

The role of the frontal ganglion and corpora cardiaca/corpora allata complex in post-feeding weight loss in adult Heliothis zea

Abstract A post-feeding diuretic response has been observed in adult Heliothis zea (Boddie) (Lepidoptera: Noctuidae). 2-day-old starved adults which were allowed to feed to repletion on a 10% (w/v) sucrose solution lost 49.1% (females) and 85.8% (males) of the weight of the ingested meal during the first hour following feeding. Ligation between head and thorax or frontal ganglionectomy, when performed immediately following feeding, each resulted in a significant and permanent reduction in this normal weight-loss. Injection of homogenates of the corpora cardiaca/corpora allata (CC/CA) complex into non-ligated insects immediately after feeding also reduced significantly the post-feeding weight-loss, but this inhibition was transient and disappeared after 1 h. Dissection and weighing of the crop from either ligated, frontal ganglionectomized, or CC/CA-injected insects confirmed the crop as the predominant site of fluid retention in each case. We suggest that a soluble antidiuretic factor from the CC/CA acts in conjunction with the frontal ganglion to control the rate of crop emptying and subsequent diuresis by regulating the volume of ingested fluid that is passed into the haemolymph from the crop/midgut.     

The effects of dietary sodium and potassium on rapid diuresis in the tsetse fly Glossina morsitans

A self-supporting colony of Glossina morsitans can be maintained by feeding flies through artificial membranes but only if a diet of defibrinated pig blood is used. The potassium concentration of pig serum was found to be higher than that of cow or rabbit serum. However, this did not affect the rate of diuresis which was lower after in vitro diets than after feeding on a live host.After feeding in vivo on rabbits, and in vitro on pig blood and cow blood, the concentrations of sodium and potassium in the haemolymph remained constant. After feeding on salt solutions and distilled water, on the other hand, it was found that the more the composition of the meal varied from that of blood serum the lower was the rate of diuresis and the more the concentrations of sodium and potassium in the haemolymph were displaced from their normal values. Large fluctuations in the concentrations of these ions resulted in the paralysis and death of the flies.The results demonstrate that the tsetse fly is able to reabsorb sodium from the primary urine produced by the Malpighian tubules during diuresis and it is suggested that this reabsorption of ions may lead to an increased circulation of water through the excretory system.     

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.     

Influence of sex and immunocastration on feed intake behavior,skatole and indole concentrations in adipose tissue of pigs

Feed intake behavior was studied between 9 weeks of age and slaughter in a total of 36 gilts, 32 immunocastrates, 33 surgically castrated barrows and 33 boars from 36 litters. Consequences for the concentration of substances contributing to off odor of pork (skatole, indole) were evaluated. Animals were kept in groups of 12 pigs of the same sex and treatment and fed ad libitum (13.4 MJ ME, 17% CP, 1.1% lysine). Individual feed intake behavior was recorded continuously by an electronic feeder. Immunocastration was carried out with two injections of Improvac with at least 4 weeks between both injections (1st: 12 to 17 weeks of age, 2nd: 19 to 21 weeks of age). Feed intake/day increased from an average of 0.91 ± 0.02 kg/day up to 3.15 ± 0.04 kg/day before slaughter. This increase was associated with a 50% reduction in the number of meals/day (from 15.8 ± 0.44 to 7.2 ± 0.29 meals/day). The larger meal sizes resulted from an increase in both, the duration of feed intake/meal and the feed intake rate (g/min). In addition, sex and treatment differences were observed: Feed intake in boars was lower than in all other groups due to a reduction in the number of meals/day and in the time spent feeding/day. In females, time spent feeding/day was quite similar to boars, but resulted from a higher number of meals of shorter duration. Barrows had a significantly higher feed intake because of a higher number of meals/day resulting in more time spent feeding/day. The feed intake rate was similar in boars, gilts and barrows and showed an increasing trend during the study, starting from about 15 g/min up to four times the amount. Immunocastration affected feed intake behavior severely, especially the meal size increased dramatically because of higher feed intake rate, which exceeded that of all other groups by 25% at the end of the study. The number of meals/day was not influenced by immunocastration and was almost identical to that of boars. Highest skatole concentrations were measured in fat of boars, whereas indole concentrations were higher in immunocastrates than in all other groups. In gilts and barrows, skatole concentrations were related to growth rate. Additionally, the feeding rate was an important factor explaining the variability in skatole/indole concentrations in adipose tissue. The physiological mechanisms however need further clarification.     

Measuring meals: structure of prandial food and water intake of rats

Attempts to understand ingestion have sought to understand the control of meals. The present study evaluated a meal definition that included prandial drinking (drinking-explicit meals). The spontaneous nocturnal intake of male Wistar rats was studied. The meal breakpoint was defined as the interval between feeding or drinking events providing the most stable estimate of meal structure. Alternative breakpoints derived from prevailing methodology, log-survivorship, or frequency histogram analysis of interfeeding intervals without respect to drinking were compared (drinking-naive meals). Threshold interfeeding intervals that accounted for drinking indirectly were evaluated as surrogate breakpoints (drinking-implicit meals). Definitions were compared by determining which criterion better conformed to predictions of satiety. Microstructural differences resulting from the definitions were also studied. Under the drinking-explicit definition, rats averaged nine or ten 13-min meals/night, during which they consumed food and water equally in duration (5-6 min) and quantity (2.3 g). Individual differences were observed in microstructure measures. Meals defined by drinking-informed, but not drinking-naive, methods were followed by the behavioral satiety sequence and by an initially low likelihood of resuming feeding that monotonically increased with time. The drinking-explicit definition uniquely revealed preprandial and postprandial correlations of similar magnitude. Under drinking-informed definitions, food restriction increased meal size, whereas drinking-naive definitions increased meal frequency. Drinking-implicit definitions provided workable approximations of meal frequency and size but inferior estimates of feeding duration, eating rate, and the preprandial correlation. Thus log-survivorship analysis is not appropriate for identifying meal breakpoints, and the consideration of drinking in meal definitions can provide a better estimate of meal structure.     

The control of diuresis in the tsetse fly Glossina austeni: a preliminary investigation of the diuretic hormone.

The rate of secretion of the Malpighian tubules of Glossina austeni is controlled by a diuretic hormone. This hormone is present in the nervous tissue of the fly together with a degradative enzyme that can be activated by boiling. It is demonstrated that the Malpighian tubules are able to destroy the diuretic hormone; they may therefore participate in the control of diuresis. The diuretic hormone appears to be a heat-stable, non-dialysable, alcohol-soluble molecule, containing amino acid, glucose and sialic acid residues.     

Role in Diuresis of a Calcitonin Receptor (GPRCAL1) Expressed in a Distal-Proximal Gradient in Renal Organs of the Mosquito Aedes aegypti (L.)

Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na⁺, Cl⁻) is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH₃₁) are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL) in the mosquito Malpighian tubules (MTs), the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH₃₁ receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH₃₁. Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs arose early in evolution.     

Feeding behaviour of artificially reared Romane lambs

A consequence of increasing litter size in sheep is that a portion of the lambs have to be reared artificially. Detailed information about the pattern of milk consumption of artificially reared lambs would help improve their management. The purpose of this study is to describe the individual and group feeding behaviour of 94 Romane artificially reared lambs from 5 to 28 days of age using an electronic automatic lamb feeder. Animals were located in four pens of 8 to 15 lambs of similar age with one teat per pen. They were fed ad libitum. In our experimental situation (group rearing, continuous lightning) on average a lamb made 1.4±0.7 visits to the teat per meal and 9.5±3 meals per day. Mean meal duration was 247±158 s and the mean daily time spent feeding was 38±25 min. The mean quantity of milk intake was 176±132 ml per meal and 1.68±0.8 l per day. With age, the number of daily meals and their duration decreased while the quantity of milk consumed per meal and per day increased. Females tended to make more visits to the teat per meal and perform more meals per day but their milk consumption per meal was lower. The feed conversion ratio was 1.36±0.2. Synchrony in feeding (group meal) was estimated as the percentage of lambs that wanted to access the teat within the same short period (relative group meal size). On average 65% of lambs in the pen wanted to access the teat within the same period, but for 35% of group meals the relative group meal size was >90%. There was no consistency in the order in which lambs accessed the teat during a group meal. Our evaluation suggested that electronic automatic lamb feeders are tools that can provide, on a large scale, data describing the feeding behaviour of artificially reared lambs. It is then possible to study factors influencing these traits in order to improve the outcome of artificially reared lambs.     

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.