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Effects of chronic obesity and weight loss on plasma ghrelin and leptin concentrations in dogs
Research in Veterinary Science 79 (2005) 169–175 www.elsevier.com/locate/rvsc
Effects of chronic obesity and weight loss on plasma ghrelin and leptin concentrations in dogs Isabelle C. Jeusette e, Johanne Detilleux b, Haruki Shibata c, Masayuki Saito d, Tsutomu Honjoh c, Agathe Delobel a, Louis Istasse a, Marianne Diez a,* a
Animal Nutrition Unit, Veterinary Faculty, B43, University of Liege, B-4000 Liege, Belgium Quantitative Genetic Unit, Veterinary Faculty, B43, University of Liege, B-4000 Liege, Belgium c Morinaga Institute of Biological Science, 2-1-1 Shimosueyoshi, Tsurumi-ku, Yokohama, Kanagawa 230-8504, Japan Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, 060-0818 Sapporo, Japan e Affinity-Petcare, 08174, Barcelona, Spain b
d
Accepted 5 November 2004
Abstract The objective of this study was to evaluate, in dogs, the effects of obesity and weight loss on plasma total ghrelin and leptin concentrations. Twenty-four Beagle dogs, 12 control lean and 12 obese dogs of both genders and aged between 1 and 9 years, were used for the experiments. Mean body weight was 12.7 ± 0.7 kg for the lean group and 21.9 ± 0.8 kg for the obese group. The trial was divided into three phases. During phase 1, all 24 Beagle dogs were fed a maintenance diet. During phase 2, the obese dogs were submitted to a weight loss protocol with a high protein-low energy diet. The weight loss protocol ended once dogs reached optimal body weight. During phase 3, the dogs that were submitted to the weight loss protocol were maintained at their optimal body weight for 6 months. Plasma total ghrelin, leptin, insulin and glucose concentrations were measured to evaluate the effects of obesity and weight loss on these parameters in dogs. Body weight, body condition score, thoracic and pelvic perimeters, and ingested food amounts were also recorded during the study. Obese dogs demonstrated a significant decrease in plasma ghrelin and a significant increase in plasma leptin and insulin concentrations when compared with control dogs. During weight loss, significant increases in plasma total ghrelin and glucose and significant decreases in plasma leptin and insulin were observed. The increase in plasma ghrelin concentrations seemed to be transient. Body weight and the morphometric parameters correlated positively with leptin concentrations and negatively with total ghrelin concentrations. These results suggest that ghrelin and leptin could play a role in dogs in the adaptation to a positive or negative energy balance, as observed in humans. 2005 Published by Elsevier Ltd. Keywords: Dog; Obesity; Leptin; Ghrelin; Insulin; Glucose; Long term body weight loss; Diet
1. Introduction Ghrelin, a 28 amino-acid gastric peptide, was identified as an endogenous ligand of the growth hormone (GH) secretagogue receptor (Kojima et al., 1999; Tomasetto et al., 2000). Ghrelin was identified in dogs by
*
Corresponding author: Fax +32 4 366 41 22. E-mail address: [email protected] (M. Diez).
0034-5288/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2004.11.012
Tomasetto et al. (2001). As in other species, the major site of ghrelin expression in dogs is the stomach and canine ghrelin presents a high degree of homology with rodent and human ghrelin. In addition to its GH-releasing properties, exogenous ghrelin stimulates food intake and adiposity in rodents (Wren et al., 2001a; Tschop et al., 2000) and in humans (Peino et al., 2000; Wren et al., 2001b). In human studies, ghrelin levels were found to decrease in cases of obesity (Tschop et al., 2001; Shiiya et al., 2002) and to increase with weight loss (Hansen
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et al., 2002). Leptin is a protein synthesized and secreted primarily by adipocytes and also by the stomach, and circulating leptin concentrations are elevated in obese humans and rodents. Plasma leptin concentrations are a good index of adiposity in dogs (Ishioka et al., 2002). Short-chain fructooligosaccharides (sc-FOS) are synthetic indigestible oligosaccharides of small length (2–4 units) which have prebiotic and dietary fibre properties. Prebiotics are non-digestible feed components that benefit the hostsÕ health by selectively stimulating the growth of one or a limited number of bacteria in the large intestine (Roberfroid, 1993). The aim of this study was to determine the effects of obesity and weight loss on total plasma ghrelin and leptin concentrations in dogs, in order to determine if ghrelin and leptin could play a role in the control of energy balance in dogs. The effects of sc-FOS supplementation during weight loss were also evaluated.
2. Materials and methods 2.1. Dogs and feeding conditions The trial was divided into three phases. 2.1.1. Phase 1 Twenty-four Beagle dogs (12 control and 12 obese dogs) aged between 1 and 9 years were used for this study. All the dogs were assessed as healthy (except for obesity) based on physical examination, normal complete blood count and serum biochemistry profile. Body condition score (BCS) was assessed according to a validated 9-point body condition scoring system (Laflamme et al., 1994). The lean control group consisted of 12 Beagle dogs, six neutered males, three intact females and three spayed females. Mean body weight (BW) was 12.7 ± 0.7 kg and mean age was 4.4 ± 0.9 years. Mean age of male and female dogs was 3.3 ± 1.5 and 5.5 ± 1.1, respectively. All dogs had a BCS of 5 on the 9-point scale. The obese group consisted of 12 obese Beagle dogs, six neutered males, three intact females and three spayed females. Mean BW was 21.9 ± 0.8 kg and mean age was 4.7 ± 0.6 years. Mean age of male and female dogs was 4.8 ± 1.2 and 4.5 ± 0.7, respectively. The BCS was 7 or 8 on the 9-point scale. Obesity had been induced by feeding a maintenance diet in large quantities over a period of 10–15 months. The dogs were chronically grossly obese for at least 1 year at the beginning of the study. All the dogs were kept in similar conditions throughout the study and were separately fed once a day a maintenance commercial extruded (dry-type) diet (Premium Croc Adult, Affinity Petcare, Spain) that was formulated to contain 24.0% crude protein, 16.1% fat, 38% starch, 2.5% crude fibre, 6% ash, and 17.32 kJ/g metabolizable
energy. For a period of 1 month, spontaneous individual energy intake was recorded daily to determine a baseline value. During this phase, control lean dogs were separately fed a fixed amount of the maintenance diet to maintain their BW while obese dogs were offered the maintenance diet in large amount during 90 min. Food left uneaten after 90 min was collected and weighed. 2.1.2. Phase 2 The obese group was divided in two similar subgroups and submitted to a weight loss protocol with two different nutritional treatments: a high protein – low energy commercial extruded (dry type) diet (Obesity Veterinary Diet, Royal Canin, France) that was formulated to contain 34.0% (w/w) crude protein, 10.0% fat, 19.3% starch, 19.8% dietary fiber, 11.2% crude fibre, 1% sc-FOS, 6% ash, 12.6 kJ/g metabolizable energy alone or the same diet supplemented with a further 2% sc-FOS (Short chain fructooligosaccharides, BeghinMeiji Industrie, France). The amount fed was reassessed each week and adjusted, if necessary, by 5% reduction of the amount offered to obtain a weekly weight loss rate of 1–2% of initial BW. The weight loss protocol was stopped once dogs had reached optimal BW as determined by a BCS of 5 on the 9-point scale. 2.1.3. Phase 3 During the last phase of the study, the optimal BW of the dogs was maintained during a 6-month period. Dogs were fed the maintenance diet again, supplemented or not with 3% sc-FOS. The amount fed was reassessed each week and adjusted, if necessary, to maintain optimal BW by a 5% increase or decrease of the amount offered. Drinking water was provided ad libitum during the entire study. Dogs were maintained in their usual kennel (two dogs in each kennel) during the whole study. Each pen was 4 · 3 m and contained a dog house. The activity level was not measured. The protocol was approved by the Animal Use and Care Advisory Committee of the University of Lie`ge. 2.2. Measurement of morphometric and blood variables Blood samples for the measurement of total ghrelin, leptin, insulin and glucose concentrations were collected once during phase 1, at the beginning and at the end of phase 2, and at the beginning and at the end (except for leptin concentrations) of phase 3. Blood from all dogs was collected between 9 and 10 AM after a 24-h fast. Plasma aliquots were frozen at 80 C in plastic tubes until assayed. Plasma total ghrelin and insulin concentrations were measured using a commercial radioimmuno assay (RIA) kits (Total ghrelin RIA kit, Linco research, MI, USA, INS-IRMA, Biosource Europe, Belgium). Two ghrelin specific RIAs have been established: one recognizes the octanoyl-modified portion and
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another the C-terminal portion of ghrelin (Hosoda et al., 2000). The kit used in this study recognized the two forms of ghrelin and not only the active acylated form. Indeed, the latter is very unstable making its measurement in stored plasma samples unreliable. Plasma glucose concentrations were determined by use of an automatic analyzer (Technicon RA 1000, Bayer, France) with commercial kit (Glucose Reagents, Technicon RA, Bayer diagnostics, France). Plasma leptin concentrations were assayed using a canine-specific ELISA method validated by Iwase et al. (2000). The within assay coefficients of variation of these blood parameters were calculated using 10 plasma samples of the same dog treated separately. BWs were recorded weekly. The BCS, thoracic and pelvic perimeters (TP, PP), excess BW and BW loss were recorded during phase 1, at the end of phase 2 and at the beginning and end of phase 3. Excess BW was expressed in % of target BW (TBW), with TBW = 0% excess BW. BW loss was expressed in % of initial BW, with a BW loss = 0 at the beginning of the trial. 2.3. Statistical analyses Values are expressed as means ± standard error of the mean (SEM). Data were normally distributed and were submitted to a univariate analysis of variance (ANOVA) for repeated measurements with group as fixed effect. The different groups were control lean dogs, obese dogs, dogs immediately and 6 months after weight loss, fed the 2 different diets supplemented or not with sc-FOS. The effect of gender was also tested. A compound symmetry or an autoregressive structure of first order (SAS Proc Mixed, SAS System Release 8.2, SAS institute Inc., Cary, NC, USA) was assumed to account for the tendency between repeated measures. A PearsonsÕ correlation analysis (SAS Proc Corr, SAS System Release 8.2, SAS institute Inc., Cary, NC, USA) was performed on the data. Differences were considered statistically significant at P < 0.05.
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low energy diet, spontaneous food intake decreased ( 47%, P < 0.001) while BW remained unchanged. Mean energy consumption to induce a weekly rate of weight loss of 1.3% was 109 ± 1.0 kcal/kg0.75 TBW, which represents a decrease of 46% (P < 0.001) when compared with the energy intake of obese dogs. Mean BW loss at the end of the weight loss protocol was 31.9%. All dogs reached TBW. Adjustments in energy intake were needed in phase 3 to maintain TBW. At the end of phase 3, mean energy consumption had to be decreased by 21% to maintain TBW (Table 1). 3.2. Ghrelin, leptin, insulin and glucose plasma concentrations When compared with control lean dogs, obese dogs had lower plasma ghrelin (P = 0.04) and higher plasma leptin (P < 0.001) and insulin (P < 0.001) concentrations. In obese dogs, ghrelin concentrations were 1.7fold lower and leptin and insulin concentrations were 5.8- and 1.9-fold higher, respectively. Once obese dogs were given the low energy diet, a leptin concentration decrease of 25% (P = 0.01) was observed while plasma ghrelin, insulin and glucose concentrations remained unchanged. During weight loss, decreases in plasma leptin ( 77%, P < 0.001) and insulin ( 43%, P = 0.02) concentrations and increases in plasma ghrelin (+163%, P = 0.001) and glucose (+17%, P < 0.001) concentrations were observed. When compared with control lean dogs, plasma glucose and ghrelin concentrations at the end of the weight loss protocol were increased by 23% (P = 0.02) and 50% (P < 0.001), respectively. Six months after the end of the weight loss protocol (end of phase 3), a decrease in plasma ghrelin ( 21%, P = 0.05) and glucose ( 7%, P = 0.03) concentrations were observed. Plasma total ghrelin concentrations were similar to those observed in control lean dogs (Table 2). 3.3. Correlation analysis
3. Results Within assay coefficients of variation of plasma total ghrelin, leptin, insulin and glucose concentrations were 5.3%, 3.6%, 5.9% and 1.4%, respectively. Results of plasma metabolites and morphometric measures are presented in Tables 1 and 2. As sc-FOS supplementation or gender resulted in no significant difference in blood metabolites, data were pooled. 3.1. Energy intake and BW Energy intake of obese dogs was higher than control lean dogs (P < 0.001). Once obese dogs were given the
When all the data were pooled, plasma total ghrelin concentrations were positively correlated with glucose concentrations (P < 0.001), BW loss (P < 0.01) and age (P = 0.04) and negatively with leptin concentrations, BW, TP, PP, BCS and excess BW (P < 0.001). Plasma leptin concentrations correlated positively with insulin concentrations, BW, TP, PP, BCS and excess BW (P < 0.001) and negatively with glucose concentrations (P = 0.05) and BW loss (P = 0.02) (Table 3). In obese dogs fed the maintenance diet, a significant positive correlation was found between leptin concentrations and BCS (r2 = 0.71, P < 0.01) or excess BW (r2 = 0.59, P = 0.04). In obese dogs fed the low energy diet, a positive correlation was found between ghrelin
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Table 1 Values (means ± standard error of the mean) of body weight, body condition score, thoracic perimeter, pelvic perimeter, excess body weight, body weight loss and ingested food amounts during phases 1, 2 and 3 of the trial BW
BCS
PT
PP
Excess BW
BW loss
Energy intake
kg
9-point scale
cm
cm
%
%
kJ/kg0.75 TBW
Phase 1: Complete and balanced diet Control 12.7 ± 0.7a Obese 21.9 ± 0.8c
5 ± 0.1a 7 ± 0.1b
55.1 ± 1.0a 71.0 ± 1.3d
45.2 ± 1.1a 64.2 ± 1.9c
0.0 ± 0.0a 48.6 ± 1.3b,1
0 ± 0a 0 ± 0a
490 ± 0a 840 ± 18c
Phase 2: Low energy diet Before weight loss 21.5 ± 0.8c After weight loss 14.8 ± 0.3b
Not measured 5 ± 0a
Not measured 58.9 ± 0.6b
Not measured 49.4 ± 1.0b
46.3 ± 1.4b 0.5 ± 0.0a
1.4 ± 2.4a 31.9 ± 1.5b
463 ± 12a 444 ± 22a
Phase 3: Complete and balanced diet Beginning 14.05 ± 0.3a,b 6 months latter 14.8 ± 0.3b
5 ± 0a 5 ± 0a
57.8 ± 0.8b 59.9 ± 0.7c
47.6 ± 1.5a,b 49.2 ± 1.6b
4.6 ± 0.1a 1.2 ± 0.0a
35.4 ± 1.4b 31.4 ± 1.8b
552 ± 33b 471 ± 34a
Units
TBW, target body weight; BCS, body condition score; TP, thoracic perimeter; PP, pelvic perimeter. a–d Values with different letters are significantly (P < 0.05) different.
Table 2 Concentrations (means ± standard error of the mean) in plasma total ghrelin, leptin, insulin and glucose during phase 1, 2 and 3 of the trial Ghrelin
Leptin
Insulin
Glucose
Units
pg/ml
ng/ml
pmol/l
mmol/l
Phase 1: Maintenance diet Control Obese
4084 ± 460b 2336 ± 150a
2.29 ± 0.43a 13.18 ± 1.93c
56.2 ± 9.0a 107.2 ± 12.7b
4.85 ± 0.12a 4.85 ± 0.18a
Phase 2: Low energy diet Before weight loss After weight loss
2950 ± 483a 5145 ± 861c
9.88 ± 1.58b 2.52 ± 0.57a
100.0 ± 11.6b 72.8 ± 6.7a,b
5.08 ± 0.07a 6.16 ± 0.10c
Phase 3: Maintenance diet Beginning After 6 months
6148 ± 652c 4855 ± 557b,c
3.00 ± 0.68a Not measured
61.1 ± 6.1a 63.4 ± 9.3a
5.93 ± 0.14b,c 5.51 ± 0.09b
a–c
Values with different letters are significantly (P < 0.05) different.
Table 3 Significant (P < 0.05) coefficients of correlation between ghrelin, leptin, insulin, glucose, morphometric data, excess body weight, body weight loss and age from all the pooled data of the whole dogs
Ghrelin Leptin Insulin Glucose BW BCS TP PP Excess BW Loss BW Age
Number of data
Ghrelin
72 60 60 60 72 60 60 60 72 72 72
0.33 NS 0.46 0.47 0.45 0.45 0.34 0.49 0.49 0.24
Leptin 0.33 0.52 0.25 0.80 0.82 0.75 0.75 0.82 0.46 NS
Insulin
Glucose
NS 0.52
0.46 NS NS
NS 0.50 0.55 0.55 0.54 0.54 NS NS
NS 0.39 NS NS 0.40 0.70 NS
BW, body weight; BCS, body condition score; TP, thoracic perimeter; PP, pelvic perimeter; NS, not significant.
concentrations and age (r2 = 0.74, P < 0.01) and between leptin and BW (r2 = 0.78, P < 0.01) or excess BW (r2 = 0.70, P = 0.01). Morphometric data were not measured at the beginning of phase 2. No signif-
icant correlation between the different parameters was found after weight loss or in control lean dogs, except a positive correlation between plasma leptin concentrations and BW (r2 = 0.57, P = 0.05).
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4. Discussion The objective of this study was, firstly, to identify, in dogs, correlations between obesity and blood parameters commonly associated with obesity in humans and secondly, to determine the effects of weight loss in obese dogs on these blood parameters. In both cases (obesity or weight loss), the effects of the diet composition as well as the relations between the different parameters, were considered. We chose to use chronically grossly obese dogs of different ages and genders to be as close as possible to field conditions. However, obese and lean groups were very similar in age and sex to allow comparison between groups. The dogs used in this study became obese by ad libitum consumption of a maintenance diet and they were grossly obese with a stable BW for at least 1 year before the beginning of the study. Many parameters (diet, food intake, age, sex, breed of dogs) were controlled to validate comparison between dogs. The effects of gender or age were not significant in this experiment; therefore data were pooled. 4.1. Obese dogs Plasma leptin and insulin concentrations were increased in obese dogs, suggesting that obese dogs, like obese humans, are leptin and insulin resistant. Obese dogs had reduced fasting plasma ghrelin concentrations, similar to what has been observed in obese humans (Tschop et al., 2001; Shiiya et al., 2002) and rodents (Ariyasu et al., 2002). These results indicate that ghrelin is downregulated as a consequence of excess dietary energy or excess energy storage. However, the values observed in lean and obese dogs were higher than values reported in humans. This could be due, in part, to the 24-h fast imposed on the dogs in our study while humans generally tend to be fasted only overnight. Human studies have focused on the role of insulin or leptin on ghrelin regulation. In obese humans, it has been suggested that the downregulation of plasma ghrelin concentrations is a consequence of elevated insulin or leptin concentrations (Tschop et al., 2001). Insulin could play a pivotal role in regulating BW through its opposite effects on plasma ghrelin and leptin concentrations (Saad et al., 2002). In the current study, plasma leptin and insulin concentrations were higher in obese dogs, but this did not significantly correlate with ghrelin concentrations. However, when data from the whole study were pooled, ghrelin concentrations correlated negatively with leptin concentrations, and insulin concentrations correlated positively with leptin concentrations. It has been hypothesized that glucose could play a role in plasma ghrelin regulation. In fasted obese rats, it has been suggested that higher glucose levels may be involved in reducing plasma ghrelin levels because sugar
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intake has been shown to do so (Tschop et al., 2000). Moreover, a study in rodents demonstrated that insulin-induced hypoglycemia stimulated ghrelin secretion and that plasma ghrelin concentrations were reduced by glucose injections in a dose-dependant fashion (Ariyasu et al., 2002). These results in rats suggest that reduced blood glucose concentrations resulted in elevated plasma ghrelin levels in fasted rats of normal weight and that high blood glucose levels should be involved in the reduced plasma ghrelin levels seen in obese animals (Ariyasu et al., 2002). This hypothesis, however, is not supported by the present study. Indeed, fasted obese dogs did not have a higher plasma glucose level despite a lower plasma ghrelin level, and weight loss resulted in increased blood glucose and ghrelin concentrations. The present study in dogs also focused on diet composition. The type of diet tested, maintenance or low energy, supplemented or not with sc-FOS, had no significant effect on plasma ghrelin, insulin or glucose concentrations in obese dogs. This is in contrast to the observations in rats (Beck et al., 2002; Lee et al., 2002). The transition to the low energy diet used in the present study resulted in lower plasma leptin concentrations in obese dogs, although BWs were not statistically different. This decrease could be due to a modification of the body composition of the dogs. Indeed, the low energy diet was a high protein, low carbohydrate diet, which could have favored the lean body mass rather than the fat body mass (Diez et al., 2002). Leptin concentrations are strongly positively correlated with body fat content in dogs (Sagawa et al., 2002) more than with BW (Ishioka et al., 2002). However, an acute change in leptin concentrations induced directly or not by a modification of the diet composition, independent of changes in body fat or an effect of the negative energy balance, could also be envisaged (Raben and Astrup, 2000). Indeed, leptin is implicated in the acute regulation of energy balance and could be one of the first parameters to change in cases of negative energy balance. 4.2. Weight loss Food restriction and consequent weight loss resulted in a significant increase in plasma ghrelin and a significant decrease in plasma leptin and insulin concentrations, which is similar to reports in humans (Hansen et al., 2002) and rodents (Ariyasu et al., 2002). These data could suggest an influence of insulin or leptin on the regulation of plasma ghrelin, as observed in obese dogs. The increase in blood glucose concentrations observed at the end of the weight loss protocol was surprising. However, glucose concentrations decreased at the end of phase 3 and were in reference ranges throughout the study. In obese dogs, an intolerance to glucose and hyperinsulinaemia have been reported (Mattheeuws
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et al., 1984) but fasting plasma glucose concentrations were normal. No effect of obesity or weight loss on glucose concentrations has been previously reported in experimental dogs (Borne et al., 1996; Diez et al., 2004). The significant increase in plasma glucose concentrations observed in this study could be due to the high protein diet fed during weight loss. Some amino-acids (alanine and serine) can promote gluconeogenesis in dogs (Belo et al., 1977). In a study testing the influence of different diets on plasma glucose concentrations, a diet containing 46% protein, 27% fat and 27% carbohydrates on energy basis, which was similar to the low energy diet used in the present study resulted in the highest glucose concentrations in fasted dogs, but the differences were not found to be significant (Belo et al., 1976). When compared with control lean dogs, plasma leptin and insulin concentrations after weight loss were within reference ranges, while plasma ghrelin concentrations were significantly higher. It could be hypothesized that ghrelin is upregulated as a result of the negative energy balance during weight loss. For the different measured parameters, no significant differences were observed after weight loss between the low energy and the maintenance diet, supplemented or not with sc-FOS. Six months after the end of the weight loss protocol, while BW was strictly controlled, plasma ghrelin concentrations decreased in 10 of 12 dogs, although means were not statistically different between the beginning and the end of BW maintenance period. Ghrelin values, while higher, were not significantly different from the control lean dogs anymore. To our knowledge, this is the first time that the long-term effects of weight loss were studied for ghrelin concentrations. Similar observations were reported in human patients with anorexia nervosa where the 2-fold increase in plasma ghrelin concentrations returned to control values after renutrition and partial BW recovery, suggesting an influence of body fat content and energy intake (Tolle et al., 2003). 4.3. Correlation analysis A significant positive correlation was found in dogs between ghrelin and glucose concentrations, BW loss or age, while a negative correlation was observed in dogs between ghrelin and leptin concentrations, BW, TP, PP, BCS or excess BW. However, it should be noted that the strength of these correlations was relatively low. The most interesting correlations to note was the negative relationship between ghrelin concentration and BW or morphometric measures, and the positive relationship between ghrelin concentration and BW loss. The negative correlation between ghrelin concentration and BW or body mass index has been previously observed in humans (Shiiya et al., 2002; Tolle et al., 2003; Tschop et al., 2001). As in dogs, a negative correlation between ghrelin and leptin concentrations was ob-
served in rats by Tschop et al. (2000) and in humans by Tschop et al. (2001). A negative correlation with insulin concentration was observed in a study on obese humans (Tschop et al., 2001). This was not, however, noted in the present study. In humans, leptin concentrations are related to subcutaneous adiposity while insulin concentrations are associated with visceral fat accumulation (Cnop et al., 2002). If these data are extrapolated to dogs, it could suggest that the regulation of ghrelin secretion in the dog may be a consequence of subcutaneous rather than visceral adiposity. Leptin concentrations were significantly positively correlated with insulin concentrations, BW, excess BW, BW loss, BCS, PP or TP. These results confirm previous studies in dogs, showing that plasma leptin concentrations correlate with BCS and body fat content in lean or obese dogs (Ishioka et al., 2002; Sagawa et al., 2002). Leptin correlated with BW has been shown in lean or obese female dogs (Sagawa et al., 2002), but not in one study in male dogs before and after weight gain (Ishioka et al., 2002).
5. Summary Obese dogs presented with lower plasma ghrelin and higher plasma leptin and insulin concentrations than lean dogs, while weight loss resulted in an increase in plasma ghrelin concentrations and in a decrease in plasma leptin and insulin concentrations. These results suggest that obese dogs are leptin and insulin resistant and that ghrelin, leptin and insulin play a role in the adaptation to a positive or negative energy balance. However, 6 months after the end of the weight loss protocol, there was no significant difference observed between control lean dogs and dogs after weight loss, despite the decreased amount of food given to control the increasing BW trend. This could suggest that, in dogs, ghrelin is more influenced by BW than by food consumption. As expected, ghrelin and leptin concentrations were correlated, negatively and positively, respectively, with morphometric measures.
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Peino, R., Baldelli, R., Rodriguez-Garcia, J., Rodriguez-Segade, S., Kojima, M., Kangawa, K., Arvat, E., Ghigo, E., Diegez, C., Casanueva, F.F., 2000. Ghrelin-induced growth hormone secretion in humans. Eur. J. Endocrinol. 143, R11–R14. Raben, A., Astrup, A., 2000. Leptin is influenced both by predisposition to obesity and diet composition. Int. J. Obes. 24, 450–459. Roberfroid, M., 1993. Dietary fiber, inulin and oligofructose: a review comparing their physiological effects. Crit. Rev. Food Sci. Nutr. 33, 103–148. Saad, M.F., Bernaba, B., Kwu, C.-M., Jinagouda, S., Fahmi, S., Kogosov, E., Boyadjian, R., 2002. Insulin regulates plasma ghrelin concentration. J. Clin. Endocrin. Metab. 87, 3997– 4000. Sagawa, M., Nakadomo, F., Honjoh, T., Ishioka, K., Saito, M., 2002. Correlation between plasma leptin concentration and body fat content in dogs. Am. J. Vet. Res. 63, 7–10. Shiiya, T., Nakazato, M., Mizuta, M., Date, Y., Mondal, M.S., Tanaka, M., Nozoe, S.-I., Hosoda, H., Kangawa, K., Matsukura, S., 2002. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J. Clin. Endocrinol. Metab. 87, 240–244. Tolle, V., Kadem, M., Bluet-Pajot, M.-T., Frere, D., Foulon, C., Bossu, C., Dardennes, R., Mounier, C., Zizzari, P., Lang, F., Epelbaum, J., Estour, B., 2003. Balance in ghrelin and leptin levels in anorexia nervosa patients and constitutionally thin women. J. Endocrin. Metab. 88, 109–116. Tomasetto, C., Wendling, C., Rio, M.-C., Poitras, P., 2001. Identification of cDNA encoding motilin related peptide/ghrelin precursor from dog fundus. Peptides 22, 2055–2059. Tomasetto, C., Karam, S.M., Ribieras, S., Masson, R., Lefebvre, O., Staub, A., Alexander, G., Chenard, M.P., Rio, M.C., 2000. Identification and characterisation of a novel gastric peptide hormone: the motilin-related peptide. Gastroenterology 119, 395– 405. Tschop, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Tschop, M., Weyer, C., Tataranni, P.A., Devanarayan, V., Ravussin, E., Heiman, M.L., 2001. Circulating ghrelin levels are decreased in human obesity. Diabetes 50, 707–709. Wren, A.M., Small, C.J., Abbott, C.R., Dhillo, W.S., Seal, L.J., Cohen, M.A., Batterham, R.L., Taheri, S., Stanley, S.A., Ghatei, M.A., Bloom, S.R., 2001a. Ghrelin causes hyperphagia and obesity in rats. Diabetes 50, 2540–2547. Wren, A.M., Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K.G., Dhillo, W.S., Ghatei, M.A., Bloom, S.R., 2001b. Ghrelin enhances appetite and increases food intake in humans. J. Endocrin. Metab. 86, 5992–5995.
Effects of chronic obesity and weight loss on plasma ghrelin and leptin concentrations in dogs Isabelle C. Jeusette e, Johanne Detilleux b, Haruki Shibata c, Masayuki Saito d, Tsutomu Honjoh c, Agathe Delobel a, Louis Istasse a, Marianne Diez a,* a
Animal Nutrition Unit, Veterinary Faculty, B43, University of Liege, B-4000 Liege, Belgium Quantitative Genetic Unit, Veterinary Faculty, B43, University of Liege, B-4000 Liege, Belgium c Morinaga Institute of Biological Science, 2-1-1 Shimosueyoshi, Tsurumi-ku, Yokohama, Kanagawa 230-8504, Japan Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, 060-0818 Sapporo, Japan e Affinity-Petcare, 08174, Barcelona, Spain b
d
Accepted 5 November 2004
Abstract The objective of this study was to evaluate, in dogs, the effects of obesity and weight loss on plasma total ghrelin and leptin concentrations. Twenty-four Beagle dogs, 12 control lean and 12 obese dogs of both genders and aged between 1 and 9 years, were used for the experiments. Mean body weight was 12.7 ± 0.7 kg for the lean group and 21.9 ± 0.8 kg for the obese group. The trial was divided into three phases. During phase 1, all 24 Beagle dogs were fed a maintenance diet. During phase 2, the obese dogs were submitted to a weight loss protocol with a high protein-low energy diet. The weight loss protocol ended once dogs reached optimal body weight. During phase 3, the dogs that were submitted to the weight loss protocol were maintained at their optimal body weight for 6 months. Plasma total ghrelin, leptin, insulin and glucose concentrations were measured to evaluate the effects of obesity and weight loss on these parameters in dogs. Body weight, body condition score, thoracic and pelvic perimeters, and ingested food amounts were also recorded during the study. Obese dogs demonstrated a significant decrease in plasma ghrelin and a significant increase in plasma leptin and insulin concentrations when compared with control dogs. During weight loss, significant increases in plasma total ghrelin and glucose and significant decreases in plasma leptin and insulin were observed. The increase in plasma ghrelin concentrations seemed to be transient. Body weight and the morphometric parameters correlated positively with leptin concentrations and negatively with total ghrelin concentrations. These results suggest that ghrelin and leptin could play a role in dogs in the adaptation to a positive or negative energy balance, as observed in humans. 2005 Published by Elsevier Ltd. Keywords: Dog; Obesity; Leptin; Ghrelin; Insulin; Glucose; Long term body weight loss; Diet
1. Introduction Ghrelin, a 28 amino-acid gastric peptide, was identified as an endogenous ligand of the growth hormone (GH) secretagogue receptor (Kojima et al., 1999; Tomasetto et al., 2000). Ghrelin was identified in dogs by
*
Corresponding author: Fax +32 4 366 41 22. E-mail address: [email protected] (M. Diez).
0034-5288/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2004.11.012
Tomasetto et al. (2001). As in other species, the major site of ghrelin expression in dogs is the stomach and canine ghrelin presents a high degree of homology with rodent and human ghrelin. In addition to its GH-releasing properties, exogenous ghrelin stimulates food intake and adiposity in rodents (Wren et al., 2001a; Tschop et al., 2000) and in humans (Peino et al., 2000; Wren et al., 2001b). In human studies, ghrelin levels were found to decrease in cases of obesity (Tschop et al., 2001; Shiiya et al., 2002) and to increase with weight loss (Hansen
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et al., 2002). Leptin is a protein synthesized and secreted primarily by adipocytes and also by the stomach, and circulating leptin concentrations are elevated in obese humans and rodents. Plasma leptin concentrations are a good index of adiposity in dogs (Ishioka et al., 2002). Short-chain fructooligosaccharides (sc-FOS) are synthetic indigestible oligosaccharides of small length (2–4 units) which have prebiotic and dietary fibre properties. Prebiotics are non-digestible feed components that benefit the hostsÕ health by selectively stimulating the growth of one or a limited number of bacteria in the large intestine (Roberfroid, 1993). The aim of this study was to determine the effects of obesity and weight loss on total plasma ghrelin and leptin concentrations in dogs, in order to determine if ghrelin and leptin could play a role in the control of energy balance in dogs. The effects of sc-FOS supplementation during weight loss were also evaluated.
2. Materials and methods 2.1. Dogs and feeding conditions The trial was divided into three phases. 2.1.1. Phase 1 Twenty-four Beagle dogs (12 control and 12 obese dogs) aged between 1 and 9 years were used for this study. All the dogs were assessed as healthy (except for obesity) based on physical examination, normal complete blood count and serum biochemistry profile. Body condition score (BCS) was assessed according to a validated 9-point body condition scoring system (Laflamme et al., 1994). The lean control group consisted of 12 Beagle dogs, six neutered males, three intact females and three spayed females. Mean body weight (BW) was 12.7 ± 0.7 kg and mean age was 4.4 ± 0.9 years. Mean age of male and female dogs was 3.3 ± 1.5 and 5.5 ± 1.1, respectively. All dogs had a BCS of 5 on the 9-point scale. The obese group consisted of 12 obese Beagle dogs, six neutered males, three intact females and three spayed females. Mean BW was 21.9 ± 0.8 kg and mean age was 4.7 ± 0.6 years. Mean age of male and female dogs was 4.8 ± 1.2 and 4.5 ± 0.7, respectively. The BCS was 7 or 8 on the 9-point scale. Obesity had been induced by feeding a maintenance diet in large quantities over a period of 10–15 months. The dogs were chronically grossly obese for at least 1 year at the beginning of the study. All the dogs were kept in similar conditions throughout the study and were separately fed once a day a maintenance commercial extruded (dry-type) diet (Premium Croc Adult, Affinity Petcare, Spain) that was formulated to contain 24.0% crude protein, 16.1% fat, 38% starch, 2.5% crude fibre, 6% ash, and 17.32 kJ/g metabolizable
energy. For a period of 1 month, spontaneous individual energy intake was recorded daily to determine a baseline value. During this phase, control lean dogs were separately fed a fixed amount of the maintenance diet to maintain their BW while obese dogs were offered the maintenance diet in large amount during 90 min. Food left uneaten after 90 min was collected and weighed. 2.1.2. Phase 2 The obese group was divided in two similar subgroups and submitted to a weight loss protocol with two different nutritional treatments: a high protein – low energy commercial extruded (dry type) diet (Obesity Veterinary Diet, Royal Canin, France) that was formulated to contain 34.0% (w/w) crude protein, 10.0% fat, 19.3% starch, 19.8% dietary fiber, 11.2% crude fibre, 1% sc-FOS, 6% ash, 12.6 kJ/g metabolizable energy alone or the same diet supplemented with a further 2% sc-FOS (Short chain fructooligosaccharides, BeghinMeiji Industrie, France). The amount fed was reassessed each week and adjusted, if necessary, by 5% reduction of the amount offered to obtain a weekly weight loss rate of 1–2% of initial BW. The weight loss protocol was stopped once dogs had reached optimal BW as determined by a BCS of 5 on the 9-point scale. 2.1.3. Phase 3 During the last phase of the study, the optimal BW of the dogs was maintained during a 6-month period. Dogs were fed the maintenance diet again, supplemented or not with 3% sc-FOS. The amount fed was reassessed each week and adjusted, if necessary, to maintain optimal BW by a 5% increase or decrease of the amount offered. Drinking water was provided ad libitum during the entire study. Dogs were maintained in their usual kennel (two dogs in each kennel) during the whole study. Each pen was 4 · 3 m and contained a dog house. The activity level was not measured. The protocol was approved by the Animal Use and Care Advisory Committee of the University of Lie`ge. 2.2. Measurement of morphometric and blood variables Blood samples for the measurement of total ghrelin, leptin, insulin and glucose concentrations were collected once during phase 1, at the beginning and at the end of phase 2, and at the beginning and at the end (except for leptin concentrations) of phase 3. Blood from all dogs was collected between 9 and 10 AM after a 24-h fast. Plasma aliquots were frozen at 80 C in plastic tubes until assayed. Plasma total ghrelin and insulin concentrations were measured using a commercial radioimmuno assay (RIA) kits (Total ghrelin RIA kit, Linco research, MI, USA, INS-IRMA, Biosource Europe, Belgium). Two ghrelin specific RIAs have been established: one recognizes the octanoyl-modified portion and
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another the C-terminal portion of ghrelin (Hosoda et al., 2000). The kit used in this study recognized the two forms of ghrelin and not only the active acylated form. Indeed, the latter is very unstable making its measurement in stored plasma samples unreliable. Plasma glucose concentrations were determined by use of an automatic analyzer (Technicon RA 1000, Bayer, France) with commercial kit (Glucose Reagents, Technicon RA, Bayer diagnostics, France). Plasma leptin concentrations were assayed using a canine-specific ELISA method validated by Iwase et al. (2000). The within assay coefficients of variation of these blood parameters were calculated using 10 plasma samples of the same dog treated separately. BWs were recorded weekly. The BCS, thoracic and pelvic perimeters (TP, PP), excess BW and BW loss were recorded during phase 1, at the end of phase 2 and at the beginning and end of phase 3. Excess BW was expressed in % of target BW (TBW), with TBW = 0% excess BW. BW loss was expressed in % of initial BW, with a BW loss = 0 at the beginning of the trial. 2.3. Statistical analyses Values are expressed as means ± standard error of the mean (SEM). Data were normally distributed and were submitted to a univariate analysis of variance (ANOVA) for repeated measurements with group as fixed effect. The different groups were control lean dogs, obese dogs, dogs immediately and 6 months after weight loss, fed the 2 different diets supplemented or not with sc-FOS. The effect of gender was also tested. A compound symmetry or an autoregressive structure of first order (SAS Proc Mixed, SAS System Release 8.2, SAS institute Inc., Cary, NC, USA) was assumed to account for the tendency between repeated measures. A PearsonsÕ correlation analysis (SAS Proc Corr, SAS System Release 8.2, SAS institute Inc., Cary, NC, USA) was performed on the data. Differences were considered statistically significant at P < 0.05.
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low energy diet, spontaneous food intake decreased ( 47%, P < 0.001) while BW remained unchanged. Mean energy consumption to induce a weekly rate of weight loss of 1.3% was 109 ± 1.0 kcal/kg0.75 TBW, which represents a decrease of 46% (P < 0.001) when compared with the energy intake of obese dogs. Mean BW loss at the end of the weight loss protocol was 31.9%. All dogs reached TBW. Adjustments in energy intake were needed in phase 3 to maintain TBW. At the end of phase 3, mean energy consumption had to be decreased by 21% to maintain TBW (Table 1). 3.2. Ghrelin, leptin, insulin and glucose plasma concentrations When compared with control lean dogs, obese dogs had lower plasma ghrelin (P = 0.04) and higher plasma leptin (P < 0.001) and insulin (P < 0.001) concentrations. In obese dogs, ghrelin concentrations were 1.7fold lower and leptin and insulin concentrations were 5.8- and 1.9-fold higher, respectively. Once obese dogs were given the low energy diet, a leptin concentration decrease of 25% (P = 0.01) was observed while plasma ghrelin, insulin and glucose concentrations remained unchanged. During weight loss, decreases in plasma leptin ( 77%, P < 0.001) and insulin ( 43%, P = 0.02) concentrations and increases in plasma ghrelin (+163%, P = 0.001) and glucose (+17%, P < 0.001) concentrations were observed. When compared with control lean dogs, plasma glucose and ghrelin concentrations at the end of the weight loss protocol were increased by 23% (P = 0.02) and 50% (P < 0.001), respectively. Six months after the end of the weight loss protocol (end of phase 3), a decrease in plasma ghrelin ( 21%, P = 0.05) and glucose ( 7%, P = 0.03) concentrations were observed. Plasma total ghrelin concentrations were similar to those observed in control lean dogs (Table 2). 3.3. Correlation analysis
3. Results Within assay coefficients of variation of plasma total ghrelin, leptin, insulin and glucose concentrations were 5.3%, 3.6%, 5.9% and 1.4%, respectively. Results of plasma metabolites and morphometric measures are presented in Tables 1 and 2. As sc-FOS supplementation or gender resulted in no significant difference in blood metabolites, data were pooled. 3.1. Energy intake and BW Energy intake of obese dogs was higher than control lean dogs (P < 0.001). Once obese dogs were given the
When all the data were pooled, plasma total ghrelin concentrations were positively correlated with glucose concentrations (P < 0.001), BW loss (P < 0.01) and age (P = 0.04) and negatively with leptin concentrations, BW, TP, PP, BCS and excess BW (P < 0.001). Plasma leptin concentrations correlated positively with insulin concentrations, BW, TP, PP, BCS and excess BW (P < 0.001) and negatively with glucose concentrations (P = 0.05) and BW loss (P = 0.02) (Table 3). In obese dogs fed the maintenance diet, a significant positive correlation was found between leptin concentrations and BCS (r2 = 0.71, P < 0.01) or excess BW (r2 = 0.59, P = 0.04). In obese dogs fed the low energy diet, a positive correlation was found between ghrelin
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Table 1 Values (means ± standard error of the mean) of body weight, body condition score, thoracic perimeter, pelvic perimeter, excess body weight, body weight loss and ingested food amounts during phases 1, 2 and 3 of the trial BW
BCS
PT
PP
Excess BW
BW loss
Energy intake
kg
9-point scale
cm
cm
%
%
kJ/kg0.75 TBW
Phase 1: Complete and balanced diet Control 12.7 ± 0.7a Obese 21.9 ± 0.8c
5 ± 0.1a 7 ± 0.1b
55.1 ± 1.0a 71.0 ± 1.3d
45.2 ± 1.1a 64.2 ± 1.9c
0.0 ± 0.0a 48.6 ± 1.3b,1
0 ± 0a 0 ± 0a
490 ± 0a 840 ± 18c
Phase 2: Low energy diet Before weight loss 21.5 ± 0.8c After weight loss 14.8 ± 0.3b
Not measured 5 ± 0a
Not measured 58.9 ± 0.6b
Not measured 49.4 ± 1.0b
46.3 ± 1.4b 0.5 ± 0.0a
1.4 ± 2.4a 31.9 ± 1.5b
463 ± 12a 444 ± 22a
Phase 3: Complete and balanced diet Beginning 14.05 ± 0.3a,b 6 months latter 14.8 ± 0.3b
5 ± 0a 5 ± 0a
57.8 ± 0.8b 59.9 ± 0.7c
47.6 ± 1.5a,b 49.2 ± 1.6b
4.6 ± 0.1a 1.2 ± 0.0a
35.4 ± 1.4b 31.4 ± 1.8b
552 ± 33b 471 ± 34a
Units
TBW, target body weight; BCS, body condition score; TP, thoracic perimeter; PP, pelvic perimeter. a–d Values with different letters are significantly (P < 0.05) different.
Table 2 Concentrations (means ± standard error of the mean) in plasma total ghrelin, leptin, insulin and glucose during phase 1, 2 and 3 of the trial Ghrelin
Leptin
Insulin
Glucose
Units
pg/ml
ng/ml
pmol/l
mmol/l
Phase 1: Maintenance diet Control Obese
4084 ± 460b 2336 ± 150a
2.29 ± 0.43a 13.18 ± 1.93c
56.2 ± 9.0a 107.2 ± 12.7b
4.85 ± 0.12a 4.85 ± 0.18a
Phase 2: Low energy diet Before weight loss After weight loss
2950 ± 483a 5145 ± 861c
9.88 ± 1.58b 2.52 ± 0.57a
100.0 ± 11.6b 72.8 ± 6.7a,b
5.08 ± 0.07a 6.16 ± 0.10c
Phase 3: Maintenance diet Beginning After 6 months
6148 ± 652c 4855 ± 557b,c
3.00 ± 0.68a Not measured
61.1 ± 6.1a 63.4 ± 9.3a
5.93 ± 0.14b,c 5.51 ± 0.09b
a–c
Values with different letters are significantly (P < 0.05) different.
Table 3 Significant (P < 0.05) coefficients of correlation between ghrelin, leptin, insulin, glucose, morphometric data, excess body weight, body weight loss and age from all the pooled data of the whole dogs
Ghrelin Leptin Insulin Glucose BW BCS TP PP Excess BW Loss BW Age
Number of data
Ghrelin
72 60 60 60 72 60 60 60 72 72 72
0.33 NS 0.46 0.47 0.45 0.45 0.34 0.49 0.49 0.24
Leptin 0.33 0.52 0.25 0.80 0.82 0.75 0.75 0.82 0.46 NS
Insulin
Glucose
NS 0.52
0.46 NS NS
NS 0.50 0.55 0.55 0.54 0.54 NS NS
NS 0.39 NS NS 0.40 0.70 NS
BW, body weight; BCS, body condition score; TP, thoracic perimeter; PP, pelvic perimeter; NS, not significant.
concentrations and age (r2 = 0.74, P < 0.01) and between leptin and BW (r2 = 0.78, P < 0.01) or excess BW (r2 = 0.70, P = 0.01). Morphometric data were not measured at the beginning of phase 2. No signif-
icant correlation between the different parameters was found after weight loss or in control lean dogs, except a positive correlation between plasma leptin concentrations and BW (r2 = 0.57, P = 0.05).
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4. Discussion The objective of this study was, firstly, to identify, in dogs, correlations between obesity and blood parameters commonly associated with obesity in humans and secondly, to determine the effects of weight loss in obese dogs on these blood parameters. In both cases (obesity or weight loss), the effects of the diet composition as well as the relations between the different parameters, were considered. We chose to use chronically grossly obese dogs of different ages and genders to be as close as possible to field conditions. However, obese and lean groups were very similar in age and sex to allow comparison between groups. The dogs used in this study became obese by ad libitum consumption of a maintenance diet and they were grossly obese with a stable BW for at least 1 year before the beginning of the study. Many parameters (diet, food intake, age, sex, breed of dogs) were controlled to validate comparison between dogs. The effects of gender or age were not significant in this experiment; therefore data were pooled. 4.1. Obese dogs Plasma leptin and insulin concentrations were increased in obese dogs, suggesting that obese dogs, like obese humans, are leptin and insulin resistant. Obese dogs had reduced fasting plasma ghrelin concentrations, similar to what has been observed in obese humans (Tschop et al., 2001; Shiiya et al., 2002) and rodents (Ariyasu et al., 2002). These results indicate that ghrelin is downregulated as a consequence of excess dietary energy or excess energy storage. However, the values observed in lean and obese dogs were higher than values reported in humans. This could be due, in part, to the 24-h fast imposed on the dogs in our study while humans generally tend to be fasted only overnight. Human studies have focused on the role of insulin or leptin on ghrelin regulation. In obese humans, it has been suggested that the downregulation of plasma ghrelin concentrations is a consequence of elevated insulin or leptin concentrations (Tschop et al., 2001). Insulin could play a pivotal role in regulating BW through its opposite effects on plasma ghrelin and leptin concentrations (Saad et al., 2002). In the current study, plasma leptin and insulin concentrations were higher in obese dogs, but this did not significantly correlate with ghrelin concentrations. However, when data from the whole study were pooled, ghrelin concentrations correlated negatively with leptin concentrations, and insulin concentrations correlated positively with leptin concentrations. It has been hypothesized that glucose could play a role in plasma ghrelin regulation. In fasted obese rats, it has been suggested that higher glucose levels may be involved in reducing plasma ghrelin levels because sugar
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intake has been shown to do so (Tschop et al., 2000). Moreover, a study in rodents demonstrated that insulin-induced hypoglycemia stimulated ghrelin secretion and that plasma ghrelin concentrations were reduced by glucose injections in a dose-dependant fashion (Ariyasu et al., 2002). These results in rats suggest that reduced blood glucose concentrations resulted in elevated plasma ghrelin levels in fasted rats of normal weight and that high blood glucose levels should be involved in the reduced plasma ghrelin levels seen in obese animals (Ariyasu et al., 2002). This hypothesis, however, is not supported by the present study. Indeed, fasted obese dogs did not have a higher plasma glucose level despite a lower plasma ghrelin level, and weight loss resulted in increased blood glucose and ghrelin concentrations. The present study in dogs also focused on diet composition. The type of diet tested, maintenance or low energy, supplemented or not with sc-FOS, had no significant effect on plasma ghrelin, insulin or glucose concentrations in obese dogs. This is in contrast to the observations in rats (Beck et al., 2002; Lee et al., 2002). The transition to the low energy diet used in the present study resulted in lower plasma leptin concentrations in obese dogs, although BWs were not statistically different. This decrease could be due to a modification of the body composition of the dogs. Indeed, the low energy diet was a high protein, low carbohydrate diet, which could have favored the lean body mass rather than the fat body mass (Diez et al., 2002). Leptin concentrations are strongly positively correlated with body fat content in dogs (Sagawa et al., 2002) more than with BW (Ishioka et al., 2002). However, an acute change in leptin concentrations induced directly or not by a modification of the diet composition, independent of changes in body fat or an effect of the negative energy balance, could also be envisaged (Raben and Astrup, 2000). Indeed, leptin is implicated in the acute regulation of energy balance and could be one of the first parameters to change in cases of negative energy balance. 4.2. Weight loss Food restriction and consequent weight loss resulted in a significant increase in plasma ghrelin and a significant decrease in plasma leptin and insulin concentrations, which is similar to reports in humans (Hansen et al., 2002) and rodents (Ariyasu et al., 2002). These data could suggest an influence of insulin or leptin on the regulation of plasma ghrelin, as observed in obese dogs. The increase in blood glucose concentrations observed at the end of the weight loss protocol was surprising. However, glucose concentrations decreased at the end of phase 3 and were in reference ranges throughout the study. In obese dogs, an intolerance to glucose and hyperinsulinaemia have been reported (Mattheeuws
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et al., 1984) but fasting plasma glucose concentrations were normal. No effect of obesity or weight loss on glucose concentrations has been previously reported in experimental dogs (Borne et al., 1996; Diez et al., 2004). The significant increase in plasma glucose concentrations observed in this study could be due to the high protein diet fed during weight loss. Some amino-acids (alanine and serine) can promote gluconeogenesis in dogs (Belo et al., 1977). In a study testing the influence of different diets on plasma glucose concentrations, a diet containing 46% protein, 27% fat and 27% carbohydrates on energy basis, which was similar to the low energy diet used in the present study resulted in the highest glucose concentrations in fasted dogs, but the differences were not found to be significant (Belo et al., 1976). When compared with control lean dogs, plasma leptin and insulin concentrations after weight loss were within reference ranges, while plasma ghrelin concentrations were significantly higher. It could be hypothesized that ghrelin is upregulated as a result of the negative energy balance during weight loss. For the different measured parameters, no significant differences were observed after weight loss between the low energy and the maintenance diet, supplemented or not with sc-FOS. Six months after the end of the weight loss protocol, while BW was strictly controlled, plasma ghrelin concentrations decreased in 10 of 12 dogs, although means were not statistically different between the beginning and the end of BW maintenance period. Ghrelin values, while higher, were not significantly different from the control lean dogs anymore. To our knowledge, this is the first time that the long-term effects of weight loss were studied for ghrelin concentrations. Similar observations were reported in human patients with anorexia nervosa where the 2-fold increase in plasma ghrelin concentrations returned to control values after renutrition and partial BW recovery, suggesting an influence of body fat content and energy intake (Tolle et al., 2003). 4.3. Correlation analysis A significant positive correlation was found in dogs between ghrelin and glucose concentrations, BW loss or age, while a negative correlation was observed in dogs between ghrelin and leptin concentrations, BW, TP, PP, BCS or excess BW. However, it should be noted that the strength of these correlations was relatively low. The most interesting correlations to note was the negative relationship between ghrelin concentration and BW or morphometric measures, and the positive relationship between ghrelin concentration and BW loss. The negative correlation between ghrelin concentration and BW or body mass index has been previously observed in humans (Shiiya et al., 2002; Tolle et al., 2003; Tschop et al., 2001). As in dogs, a negative correlation between ghrelin and leptin concentrations was ob-
served in rats by Tschop et al. (2000) and in humans by Tschop et al. (2001). A negative correlation with insulin concentration was observed in a study on obese humans (Tschop et al., 2001). This was not, however, noted in the present study. In humans, leptin concentrations are related to subcutaneous adiposity while insulin concentrations are associated with visceral fat accumulation (Cnop et al., 2002). If these data are extrapolated to dogs, it could suggest that the regulation of ghrelin secretion in the dog may be a consequence of subcutaneous rather than visceral adiposity. Leptin concentrations were significantly positively correlated with insulin concentrations, BW, excess BW, BW loss, BCS, PP or TP. These results confirm previous studies in dogs, showing that plasma leptin concentrations correlate with BCS and body fat content in lean or obese dogs (Ishioka et al., 2002; Sagawa et al., 2002). Leptin correlated with BW has been shown in lean or obese female dogs (Sagawa et al., 2002), but not in one study in male dogs before and after weight gain (Ishioka et al., 2002).
5. Summary Obese dogs presented with lower plasma ghrelin and higher plasma leptin and insulin concentrations than lean dogs, while weight loss resulted in an increase in plasma ghrelin concentrations and in a decrease in plasma leptin and insulin concentrations. These results suggest that obese dogs are leptin and insulin resistant and that ghrelin, leptin and insulin play a role in the adaptation to a positive or negative energy balance. However, 6 months after the end of the weight loss protocol, there was no significant difference observed between control lean dogs and dogs after weight loss, despite the decreased amount of food given to control the increasing BW trend. This could suggest that, in dogs, ghrelin is more influenced by BW than by food consumption. As expected, ghrelin and leptin concentrations were correlated, negatively and positively, respectively, with morphometric measures.
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