• View in gallery
    Figure 1—

    Mean ± SEM plasma ghrelin concentrations in 9 Beagles before (–90 and 0 minutes) and after (30, 60, 90, 150, 210, 300, and 420 minutes) ingestion of a meal at 10 AM (A), after withholding of food for 1 day (B), after withholding of food for 3 days (C), and before and after ingestion of a meal at 10 AM the day after the 3-day food-withholding period (D).

  • View in gallery
    Figure 2—

    Mean ± SEM plasma GH concentrations in the same dogs as in Figure 1. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Mean ± SEM plasma glucose concentrations in the same dogs as in Figure 1. See Figure 1 for key.

  • View in gallery
    Figure 4—

    Mean ± SEM plasma insulin concentrations in the same dogs as in Figure 1. See Figure 1 for key.

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Effects of food intake and food withholding on plasma ghrelin concentrations in healthy dogs

Sofie F. BhattiDepartment of Small Animal Medicine and Clinical Biology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.

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Leo J. HoflandDepartment of Internal Medicine, Division of Endocrinology, Erasmus University Medical Centre, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

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Peter M. van KoetsveldDepartment of Internal Medicine, Division of Endocrinology, Erasmus University Medical Centre, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

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Luc M. Van HamDepartment of Small Animal Medicine and Clinical Biology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.

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Luc DuchateauDepartment of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.

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Jan A. MolDepartment of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 8, NL-3508 TD, Utrecht, The Netherlands.

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Aart Jan van der LelyDepartment of Internal Medicine, Division of Endocrinology, Erasmus University Medical Centre, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.

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Hans S. KooistraDepartment of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 8, NL-3508 TD, Utrecht, The Netherlands.

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Abstract

Objective—To investigate the physiologic endocrine effects of food intake and food withholding via measurement of the circulating concentrations of acylated ghrelin, growth hormone (GH), insulin–like growth factor-I (IGF-I), glucose, and insulin when food was administered at the usual time, after 1 day's withholding, after 3 days' withholding and after refeeding the next day in healthy Beagles.

Animals—9 healthy Beagles.

Procedures—Blood samples were collected from 8:30 AM to 5 PM from Beagles when food was administered as usual at 10 AM, after 1 day's withholding, after 3 days' withholding, and after refeeding at 10 AM the next day.

Results—Overall mean plasma ghrelin concentrations were significantly lower when food was administered than after food withholding. Overall mean plasma GH and IGF-I concentrations did not differ significantly among the 4 periods. Circulating overall mean glucose and insulin concentrations were significantly higher after refeeding, compared with the 3 other periods.

Conclusions and Clinical Relevance—In dogs, food withholding and food intake were associated with higher and lower circulating ghrelin concentrations, respectively, suggesting that, in dogs, ghrelin participates in the control of feeding behavior and energy homeostasis. Changes in plasma ghrelin concentrations were not associated with similar changes in plasma GH concentrations, whereas insulin and glucose concentrations appeared to change reciprocally with the ghrelin concentrations.

Abstract

Objective—To investigate the physiologic endocrine effects of food intake and food withholding via measurement of the circulating concentrations of acylated ghrelin, growth hormone (GH), insulin–like growth factor-I (IGF-I), glucose, and insulin when food was administered at the usual time, after 1 day's withholding, after 3 days' withholding and after refeeding the next day in healthy Beagles.

Animals—9 healthy Beagles.

Procedures—Blood samples were collected from 8:30 AM to 5 PM from Beagles when food was administered as usual at 10 AM, after 1 day's withholding, after 3 days' withholding, and after refeeding at 10 AM the next day.

Results—Overall mean plasma ghrelin concentrations were significantly lower when food was administered than after food withholding. Overall mean plasma GH and IGF-I concentrations did not differ significantly among the 4 periods. Circulating overall mean glucose and insulin concentrations were significantly higher after refeeding, compared with the 3 other periods.

Conclusions and Clinical Relevance—In dogs, food withholding and food intake were associated with higher and lower circulating ghrelin concentrations, respectively, suggesting that, in dogs, ghrelin participates in the control of feeding behavior and energy homeostasis. Changes in plasma ghrelin concentrations were not associated with similar changes in plasma GH concentrations, whereas insulin and glucose concentrations appeared to change reciprocally with the ghrelin concentrations.

In 1999, Kojima et al1 reported the isolation of ghrelin from the stomach of rats.The presence of ghrelin in the stomach has been confirmed in humans, cows, pigs, dogs, and horses.2–4 Ghrelin acts as the ligand for the GH secretagogue receptor type 1a and is able to stimulate GH secretion from pituitary gland cells in many species such as rats, humans, goats, dogs, and fish.1–3,5-7 The mature ghrelin molecule contains 28 amino acids, and its third serine residue is acylated by n-octanoic acid, which is essential for binding to the GH secretagogue receptor type 1a, for the GH-releasing capacity of ghrelin, and most likely for its other endocrine actions.1,8

Through activation of pathways distinct from those leading to GH secretion, ghrelin also acts as a potent orexigenic peptide.9–11 In rodents, ghrelin causes weight gain by increasing food intake and reducing fat catabolism.9,11 Ghrelin is the only known circulating orexigen and has a potency similar to that of NPY. This appetite-stimulating effect appears to be mediated, at least in part, through activation of NPY or AGRP neurons in the hypothalamic arcuate nucleus, 94% of which express the GH secretagogue receptor.12 Neuropeptide Y and AGRP are thought to be mediators of the ghrelin-induced increased food intake because antagonism of either NPY or AGRP signaling in the brain attenuates the orexigenic potency of injected ghrelin.9,13,14

Ghrelin may also play a role in meal initiation in humans because the concentration of this peptide increases immediately prior to a meal and decreases after eating.15 Also, in sheep and cows, preprandial ghrelin surges occur as many times per day as meals are provided.16–18 These results indicate that ghrelin secretion may be a trigger for endogenous appetite signals.

Consistent with this, circulating ghrelin concentrations are increased in anorexia and cachexia and reduced in obesity.19–21 These changes are opposite to those induced by leptin, an adipocyte-derived hormone that reduces appetite and increases energy expenditure in animal models. Thus, both ghrelin and leptin reflect the metabolic balance and may drive neuroendocrine and metabolic responses to changes in nutritional status.22,23

Ghrelin production in the stomach is not only stimulated by fasting, but is also related to glucose and insulin metabolism. In humans, oral and IV glucose administration decreases plasma ghrelin concentrations, whereas lipids or high-fat diets suppress postprandial ghrelin concentrations less effectively.24,25 Additionally, in humans, the ghrelin response to a highcarbohydrate meal is related to insulin secretion.26

There are only a few reports27–29 on the role of ghrelin in dogs, a carnivorous animal with an evolutionary background of being able to cope with long periods of starvation. However, modern domestic dogs do not need to endure starvation, but rather a food surplus that often leads to obesity.

The purpose of the study reported here was to investigate the physiologic endocrine effects of food intake and food withholding via measurement of the circulating concentrations of ghrelin, GH, IGF-I, glucose, and insulin when food was administered at the usual time, after 1 day's withholding, after 3 days' withholding, and after refeeding the next day, in healthy Beagles.

Materials and Methods

Dogs—Nine 5-year-old ovariohysterectomized female Beagles were used. Dogs received a commercial dog fooda (20 g/kg of bodyweight) at 10 AM each day and were given water ad libitum. The mean body weight of the dogs was 12 kg (range, 9.5 to 14.0 kg). The dogs were accustomed to the laboratory environment and procedures such as collection of blood samples. This study was approved by the Ethics Committee of the Faculty of Veterinary Medicine, Ghent University.

Study design and blood sample collection—Dogs were randomly assigned to 3 groups. For the second and third group, the study started 1 and 2 days, respectively, after the first group. All dogs were put on a food-intake–food-with-holding regimen for 5 consecutive days. On day 1, food was given as usual at 10 AM; on days 2 through 4, food was with-held; and on day 5, the dogs were refed at 10 AM.

Blood samples were collected via jugular venipuncture on days 1 (period 1), 2 (period 2), 4 (period 3), and 5 (period 4) at 8:30 AM, 10 AM, 10:30 AM, 11 AM, 11:30 AM, 12:30 PM, 1:30 PM, 3 PM, and 5 PM. In these blood samples, the plasma or serum concentrations of acylated ghrelin, GH, glucose, and insulin were determined. Plasma IGF-I concentrations were determined only in the blood samples collected at 8:30 AM and 10:00 AM.

For determination of plasma ghrelin concentrations, the collection procedures and storage were carried out in accordance with the protocol supplied by the manufacturer of the test kit.b Blood samples were immediately transferred to icechilled EDTA-coated tubes and centrifuged at 3,500 × g at 4°C for 10 minutes. For each milliliter of plasma, 50 μL of 1N HCl and 10 μL of phenylmethylsulfonyl fluoride was added. Plasma was immediately stored at −25°C until analysis.

For determination of plasma GH and IGF-I concentrations, the blood samples were immediately transferred to icechilled EDTA-coated tubes and centrifuged at 4°C for 10 minutes. Plasma was stored at −25°C until assayed.

Blood samples for measurement of plasma glucose concentrations were collected in sodium fluoride tubes, and those for the measurement of serum insulin concentrations were collected in tubes with clot activator.

Hormone analyses—Plasma ghrelin concentrations were measured with a human ghrelin RIA kit.b This assay specifically measures biologically active ghrelin.30 Because this RIA is for use with rat and human plasma, Yokoyama et al29 verified that dog plasma contained suitable matrices by establishing that the RIA technique detected rat and human ghrelin with equal accuracy in dog plasma samples that had been spiked with human or rat ghrelin. Values of ghrelin measured with the human ghrelin RIA kit after serial dilutions of various dog samples revealed good linearity, indicating that in this assay, canine ghrelin has immunochemical properties similar to those of human ghrelin standards. The sensitivity of the assay was 10 ng/L.

Plasma GH concentrations were measured with a commercially available RIA for porcine and canine GH.c The intra-assay coefficient of variation was 7.6% at a plasma concentration of 4.4 μg/L. Sensitivity of the assay was 1 μg/L.

Total plasma IGF-I concentrations were measured after acid ethanol extraction to remove interfering IGF-binding proteins. Plasma IGF was extracted by use of a mixture of 87.5% (vol/vol) ethanol and 12.5% 2M formic acid. Tubes containing 100 μL of plasma and 400 μL of the ethanol–formic acid mixture were mixed thoroughly and incubated for 30 minutes at 22°C. After centrifugation for 30 minutes at 5,500 Xg at 4°C, a 50-μL aliquot of the supernatant was diluted 1:50 with assay buffer containing 63mM Na2HPO4 (pH 7.4), 13mM Na2EDTA, and 0.25% (wt/vol) bovine serum albumin. The extraction efficiency amounted to 92.5 ± 5.7%. Plasma IGF-I concentrations were measured with a heterologous RIA validated for dogs.31 The intra-assay coefficient of variation was 8.6% at a plasma concentration of 100 μg/L. Sensitivity of the assay was 10 μg/L. Insulin–like growth factor-1 antiserum AFP4892898 and human IGF-I for iodination were obtained.d

Serum immunoreactive insulin concentrations were measured by use of immunoradiometric assay. The 2-site immunoradiometric assay methode had an intra-and interassay coefficient of variation of 4.5% and 4.7%, respectively. Serial dilutions of canine serum yielded values parallel to the standard curve of human insulin. Sensitivity of the assay was 7 pmol/L.

Statistical analyses—Changes in circulating concentrations of ghrelin, GH, glucose, and insulin were analyzed with a mixed model with dog as random effect (repeated measures ANOVA with compound symmetry structure) and period (1 through 4), blood sampling times (8:30 AM, 10 AM, 10:30 AM, 11 AM, 11:30 AM, 12:30 PM, 1:30 PM, 3 PM, and 5 PM), and their interaction as categoric fixed effects. Mean concentrations over the different sampling points (from −90 to 420 minutes and denoted by overall mean concentration) and concentrations at the different sampling points (–90, 0, 30, 60, 90, 150, 210, 300, or 420 minutes and denoted by time-specific mean concentration) were compared, with mean referring to the mean of the different dogs. Periods 1 through 4 were compared pairwise by use of the Tukey multiple comparisons technique at a global significance level of 5%. Plasma IGF-I concentrations among the 4 periods were compared by use of 1-way ANOVA. All values are expressed as mean ± SEM.f

Results

Overall mean plasma ghrelin concentration when food was given at day 1 (152 ± 34 ng/L) was significantly (P = 0.005) lower than after food was withheld for 1 day (181 ± 42 ng/L). Overall mean plasma ghrelin concentration after refeeding the day after food was withheld for 3 days (143 ± 32 ng/L) was also significantly lower than after food was withheld for 1 day (181 ± 42 ng/L; P = 0.001) and after food was withheld for 3 days (183 ± 53 ng/L; P = 0.009). Time-specific mean plasma ghrelin concentrations decreased shortly after feeding, but this decline did not reach significance. Additionally, time-specific mean plasma ghrelin response was not significantly different among the 4 time periods (Figure 1). There was considerable interindividual variation in the pre- and postfeeding changes for time-specific plasma ghrelin concentration.

Figure 1—
Figure 1—

Mean ± SEM plasma ghrelin concentrations in 9 Beagles before (–90 and 0 minutes) and after (30, 60, 90, 150, 210, 300, and 420 minutes) ingestion of a meal at 10 AM (A), after withholding of food for 1 day (B), after withholding of food for 3 days (C), and before and after ingestion of a meal at 10 AM the day after the 3-day food-withholding period (D).

Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1557

Overall mean plasma GH concentrations when food was given at day 1, after food was withheld for 1 day, after food was withheld for 3 days, and after refeeding the next day (1.7 ± 0.3 μg/L, 1.5 ± 0.2 μg/L, 1.4 ± 0.3 μg/L, and 1.5 ± 0.3 μg/L, respectively) did not differ significantly. Time-specific mean plasma GH concentration differed significantly (P < 0.001) over time, with a maximum concentration at 0 minutes. Time-specific mean plasma GH concentration did not change significantly over time among the 4 time periods (Figure 2). No significant differences were detected in overall mean plasma IGF-I concentrations among dogs given food at day 1 (63 ± 9 μg/L), after food was withheld for 1 day (60 ± 8 μg/L), after food was withheld for 3 days (53 ± 7 μg/L), and after refeeding the next day (47 ± 6 μg/L).

Figure 2—
Figure 2—

Mean ± SEM plasma GH concentrations in the same dogs as in Figure 1. See Figure 1 for key.

Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1557

Overall mean plasma glucose concentrations when food was given at day 1 (4.5 ± 0.1 mmol/L), after food was withheld for 1 day (4.6 ± 0.1 mmol/L), and after food was withheld for 3 days (4.5 ± 0.1 mmol/L) were significantly (P < 0.001) lower, compared with this concentration after refeeding the day after withholding food for 3 days (5.0 ± 0.2 mmol/L) (Figure 3). Overall mean serum insulin concentrations when food was given at day 1 (28 ± 5 U/L) and after refeeding the day after withholding food for 3 days (27 ± 4 U/L) were significantly (P < 0.001) higher than after withholding food for 1 day (14 ± 2 U/L) and 3 days (12 ± 2 U/L; Figure 4). Time-specific mean plasma glucose and serum insulin concentrations differed significantly (P < 0.001) over time. In addition, the course of these concentrations over time (ie, from −90 to 420 minutes) differed significantly (P < 0.001) among the 4 sampling periods.

Figure 3—
Figure 3—

Mean ± SEM plasma glucose concentrations in the same dogs as in Figure 1. See Figure 1 for key.

Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1557

Figure 4—
Figure 4—

Mean ± SEM plasma insulin concentrations in the same dogs as in Figure 1. See Figure 1 for key.

Citation: American Journal of Veterinary Research 67, 9; 10.2460/ajvr.67.9.1557

Discussion

The present study revealed a significant difference in overall mean plasma ghrelin concentrations between samples obtained after food intake versus food withholding, characterized by lower plasma ghrelin concentrations when a meal was administered and higher plasma ghrelin concentrations during food withholding. These findings were consistent with those in other species. For example, in rodents, circulating ghrelin concentrations increase with food with-holding and are suppressed within minutes by refeeding or enteral infusions of nutrients but not water.10,13 These observations suggest that ghrelin may play an important role in controlling feeding behavior and energy homeostasis.

The higher plasma ghrelin concentrations during food withholding versus after feeding may be consistent with a physiologic role for this hormone in increasing appetite and the initiation of food intake. Similar to the situation in rodents, circulating ghrelin concentrations in humans are rapidly suppressed by food intake, and 24-hour plasma ghrelin analysis reveals marked preprandial increases and postprandial decreases associated with every meal.15 Although mealtime hunger is a common daily experience in humans and other animals, the molecular determinants of this sensation remain incompletely understood. Several observations, mostly from nonhuman studies, indicate that ghrelin contributes to the sensation of hunger and participates in meal initiation. Most circulating ghrelin is produced by the stomach and duodenum, organs well positioned to detect recently ingested food.1,2 Despite being produced peripherally, ghrelin acts centrally to stimulate food intake.10,11 The orexigenic actions of ghrelin are rapid and short-lived, increasing both food intake13 and gastric acid secretion32 within 20 minutes after intraperitoneal injection, a time course that is consistent with a role in meal initiation. Exogenous ghrelin triggers eating when administered at times of minimal spontaneous food intake.11 Finally, the most clearly documented targets of ghrelin action in the brain are the hypothalamic neurons that cosecrete the orexigens, NPY, and AGRP.20 These neuropeptides are implicated in central regulation of meal initiation because their concentration increases at times of maximal spontaneous food intake in rodents, whereas that of other neuropeptides involved in energy balance remain fairly constant throughout the day.33

With regard to plasma ghrelin concentration in the present study, neither a significant time effect nor significant interactions among time and the 4 periods was observed. Nevertheless, on day 1, the highest time-specific plasma ghrelin concentrations were detected immediately before feeding. It is possible that this non-significant preprandial increase occurred as an anticipatory response to feeding because the dogs had been fed at the same time of the day for several years. That a psychological factor might have played a role in the present study is further supported by the observation that, after a few days of withholding of food, the highest mean time-specific plasma ghrelin concentration was observed not before food administration, but immediately after administration of food on day 5 (this administration of food could be considered, from the dogs' standpoint, unexpected). Similarly, Sugino et al16 reported that psychological factors (ie, expectation of food administration) can stimulate ghrelin secretion just before feeding in sheep. This may be part of a conditioned emotional response. It is well-known that secretion of saliva and gastric acid preceding food intake is induced by a conditioned emotional response through the stimulation of the vagal nerve.34 In this respect, ghrelin secretion may be induced by the vagal neural system in the same manner as the secretion of saliva and gastric acid.

As in humans and rodents, mean time-specific plasma ghrelin concentrations decreased after food intake in dogs, but this decrease did not reach significance. However, in another study29 in dogs, plasma ghrelin concentrations decreased significantly after eating. The results of the present study did not reveal that feeding resulted in a significantly lower overall mean plasma ghrelin concentration, compared with food withholding in dogs. The mechanisms by which nutrients suppress ghrelin concentrations are beginning to be elucidated. Absorbed nutrients are thought to be the most likely mediators of the postprandial decrease in plasma ghrelin concentrations in rodents.10 Ingested nutrients suppress ghrelin release in rats and humans with the efficacy of carbohydrates greater than that of proteins greater than that of lipids.35 Surprisingly, foodrelated ghrelin suppression does not require luminal nutrient exposure in the stomach or duodenum, the principal sites of ghrelin production.35 Instead, signals mediating this response originate more distally in the intestines and from postabsorptive events. In addition to nutrients, changes in plasma insulin concentrations, intestinal osmolarity, and enteric neural signaling probably play a role, whereas gastric distension, vagal nerve activity, and glucagon-like peptide-1 are not required.36,37

There was considerable interindividual variation in mean time-specific plasma ghrelin concentrations in the dogs of this study, as has been reported in humans.15,30 Interestingly, a strong correlation in plasma ghrelin concentrations has been found in individuals when plasma ghrelin is measured on 1 day and then again 1 year later; however, there may be a large variation in plasma ghrelin concentrations among individuals.30 This suggests that the variation in plasma ghrelin concentrations is mainly determined by the variation among individuals and less by intra-individual factors. Also, a large heterogeneity was found in the prefood surge and postfood decrease in ghrelin concentrations among dogs, indicating that not only basal ghrelin concentrations, but also the ghrelin responses to food or lack of food vary widely among individuals.

In the present study, mean time-specific plasma GH concentrations increased just before the time when feeding normally occurred. Also, in cows, a single GH surge during feeding has been detected.38 The 2 principal hypothalamic regulators of GH secretion, GH-releasing hormone and somatostatin, do not seem to be responsible for the increase in circulating GH concentrations associated with feeding.39 Because ghrelin is also a potent GH-releasing peptide, it can be hypothesized that a preprandial increase in circulating ghrelin concentrations may be responsible for the preprandial increase in GH secretion. Indeed, some studies in humans,15 goats, and sheep16,17 reveal that a preprandial increase in plasma ghrelin concentration is associated with a GH surge. Results of the present study, however, do not provide evidence for such a relationship in dogs because the significant preprandial GH surge was not associated with a significant preprandial ghrelin surge. In contrast to the overall mean plasma ghrelin profiles, the overall mean plasma GH profiles did not differ significantly in the fed state, compared with the food-withheld state. In addition, overall mean plasma IGF-I concentrations did not differ among the several food-withholding regimens. Similar to the situation in the dogs of the present study, a link between plasma ghrelin concentrations and plasma GH concentrations has not been detected in cows.18

Overall mean plasma profiles of ghrelin and over all mean profiles of insulin and glucose changed reciprocally after feeding and food withholding in the dogs reported here. Although overall mean plasma ghrelin concentrations were significantly lower after feeding than after food withholding, the opposite was true for overall mean serum insulin concentrations. These findings are in agreement with a study15 in humans, in which plasma ghrelin concentrations changed in a manner that was opposite to the changes in plasma insulin concentrations. The observations that both ghrelin and insulin are involved in the physiologic response to food intake as well as in body weight regulation and that they have reciprocal 24-hour profiles15 raises the question whether insulin regulates ghrelin in a negative feedback manner or vice versa. The former hypothesis has been investigated by many groups.40,41 Taken together, these studies40,41 reveal that although insulin can suppress ghrelin release when administered in supraphysiologic doses or at high concentrations (within reference range) for prolonged periods, physiologic concentrations of insulin do not appear to regulate ghrelin release.42,43 It has also been suggested that ghrelin may act as a counter regulatory hormone that blocks insulin secretion and insulin action to maintain blood glucose concentrations.42,44 Indeed, several studies45–47 reveal that ghrelin can inhibit glucose-mediated insulin secretion in vitro and in vivo. Similarly, exogenous ghrelin administration decreases circulating insulin concentrations in mice46 and humans.48 Recently, a novel ghrelin-producing pancreatic islet cell has been identified, the ε-cell.49 These cells are derived from the same progenitors as are the 4 classical islet cell types (which produce insulin, glucagon, somatostatin, and pancreatic polypeptide) and can replace the other islet cells when the latter are eliminated by, for example, genetic deletion of vital transcription factors. Because ghrelin is highly expressed in the fetal pancreas (6 to 7 times more than in the stomach),50 it may participate in pancreatic islet development. Preliminary evidence also suggests that ghrelin has paracrine effects on insulin secretion in adults. If ghrelin derived from the pancreatic islets, rather than from the gastrointestinal tract, is the more critical regulator of insulin release, this raises the interesting possibility of an intra-islet ghrelin and insulin glucoregulatory axis.

In dogs, food withholding and food intake were associated with higher and lower circulating ghrelin concentrations, respectively, suggesting that in this species ghrelin participated in the control of feeding behavior and energy homeostasis. The changes in plasma ghrelin concentrations were not associated with similar changes in plasma GH concentrations, whereas circulating insulin and glucose concentrations appeared to change reciprocally with the ghrelin concentration.

ABBREVIATIONS

GH

Growth hormone

NPY

Neuropeptide Y

AGRP

Agouti gene-related peptide

IGF

Insulin–like growth factor

RIA

Radioimmunoassay

a.

Science Diet lamb meal and rice adult dog food, Hill's Pet Nutrition Inc, Topeka, Kan.

b.

Ghrelin (active) RIA kit, Linco Research, St Louis, Mo.

c.

PGH-46HK, Linco research, Saint Charles, Miss.

d.

National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, Calif.

e.

INS-IRMA, BioSource Europe SA, Nivelles, Belgium.

f.

SAS, version 9.1, SAS Institute Inc, Cary, NC.

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Contributor Notes

The authors thank E. De Wolf, G. De Clercq, S. Bruggeman, J. Wolfswinkel, C.P.M. Sprang, and F. Riemers for technical assistance.

Address correspondence to Dr. Bhatti.