Obesity is one of the most common nutritional disturbances in companion dogs, and its prevalence has been increasing recently.1,2 Obesity is known to be a risk factor for diabetes,3 orthopedic disorders,1 and cardiovascular diseases1 in dogs. In addition, it has been reported that prevention of the development of obesity extends the life span of dogs.4 Because numerous underlying factors are associated with obesity,5 it is necessary to understand the pathophysiologic processes involved to prevent or treat obesity in dogs.
Leptin has been isolated as a product of the ob gene6 and modulates food intake and energy consumption in mammals.7 Previous investigations have revealed that blood leptin concentration is positively correlated with body fat storage in humans,8 rodents,9,10 and dogs.11 Obese animals have higher blood leptin concentrations, and leptin seems to have less of an effect in terms of decreasing food intake and body weight in those animals, suggesting the presence of leptin resistance.9 It is suspected that leptin resistance may be associated with development of obesity12; however, to our knowledge, there are no reports on the cause of leptin resistance in dogs.
Leptin is released from adipose tissue into the peripheral circulation and binds to leptin receptors in the hypothalamus by crossing the BBB.13,14 Decreased efficacy of leptin transport into the brain has been suggested as a cause of leptin resistance in humans15 and rodents16,17 because the CSF leptin concentrations of obese individuals are disproportionately high, compared with their blood leptin concentrations. In addition, intracerebroventricular administration of leptin decreases food intake and body weight in obese rodents, and peripheral administration does not.16,18 However, the mechanisms of leptin resistance have not been definitively elucidated.
Results of investigations of the cause of leptin resistance should contribute to understanding the pathophysiology of obesity in dogs. The purpose of the study of this report was to evaluate postprandial changes in the leptin concentration of CSF in dogs during development of obesity. To this end, postprandial changes in CSF leptin concentrations and the relationship between CSF leptin concentrations and serum leptin concentrations were evaluated in dogs during development of diet-induced obesity. Our intent was to provide new information regarding the pathogenesis of leptin resistance in obese dogs.
Materials and Methods
Animals—Four thin, sexually intact male Beagles (5 to 7 years old) that were owned by the Faculty of Applied Biological Sciences, Gifu University were used in the study. Except for their thin body condition, all dogs were assessed as clinically normal on the basis of the results of physical examination, CBC, and serum biochemical analyses. To induce weight gain, dogs were allowed free access to an extruded commercially available high-fat fooda that was formulated for dogs. Dogs were evaluated before (thin body condition) and at 7 (optimal body condition) and 17 weeks after (obese body condition) induction of weight gain. For each body condition stage, the degree of obesity was determined by use of an assigned body condition score on a 9-point scale19 and measurement of the fat area at the level of the third lumbar vertebra via computed tomography (method had been previously validated in dogs20). Also, before starting the experiments at each stage, body weight, height, and body length were recorded. The study was conducted in a manner consistent with the Gifu University Guidelines for Animal Experimentation.
Experimental protocol—Each dog was evaluated in fed and unfed states for each body condition; there was a 1-week washout period between assessments. Food was withheld from the dogs for 24 hours prior to any of the assessments. For assessments in the fed state, dogs were provided a commercially available extruded foodb formulated for dogs (617.5 kJ/kg of body weight0.67)21 at 9 AM. For assessments in the unfed state, the dogs did not receive any food. Blood and CSF samples were collected at 8 AM (baseline leptin concentrations), 4 PM, and 10 PM for measurement of serum and CSF leptin concentrations. The sampling time points were chosen on the basis of data obtained in a previous study22 in dogs, which identified an increased plasma leptin concentration postprandially. For sample collection, dogs were sedated by administration of medetomidinec (0.02 mg/dog, IV). Cerebrospinal fluid samples were collected via cisternal puncture with a 23-gauge needle, and blood samples were collected from a saphenous vein. Immediately after sample collection, dogs were administered atipamezoled (0.01 mg/dog, IV). Samples of CSF and serum were stored at −30°C until assayed.
Hormonal assays—For each sample, CSF was added to bovine serum albumine (final concentration, 20 mg of CSF/mL) and concentrated by use of a centrifugal filter unit.f Before and after concentration, albumin concentrations were measured by use of a dry chemistry methodg as an indicator of the concentration rates. Leptin concentrations in concentrated CSF and serum samples were determined by use of an ELISA that included an anticanine leptin antibody.23 The CSF leptin concentrations before concentration were calculated from the concentration rates. Analysis of the calibration curve of the CSF leptin and albumin concentrations revealed good linearity (r = 0.993). To assess the efficacy of leptin transport across the BBB, the CSFL:SL ratio was calculated.
Statistical analysis—Cerebrospinal fluid and serum leptin concentrations are expressed as means ± SEM. Differences between values in the fed and unfed states and values before and during experiments were determined by use of the paired t test. Differences among body conditions were determined by use of a 2-way ANOVA with the Fisher protected least significant difference test. For the fed state, correlations between fat area and the combined data for each variable in the thin, optimal, and obese body conditions were determined by use of the Pearson correlation coefficient test. Values of P < 0.05 were considered significant.
Results
Changes in body condition—During induction of weight gain, changes in body condition were recorded (Table 1). There was a 30% increase in body weight between the thin and optimal body conditions and between the optimal and obese body conditions. There were 8- and 4-fold increases in the fat area at L3 between the thin and optimal body conditions and between the optimal and obese body conditions, respectively. Compared with the thin body condition, body length increased slightly in the obese body condition. Height did not change during induction of weight gain.
Mean ± SEM body weight, height, body length, body condition score, and fat area at the level of the third lumbar vertebra (determined via computed tomography) in 4 dogs in thin, optimal, and obese body conditions.
| Variable | Body condition | ||
|---|---|---|---|
| Thin | Optimal | Obese | |
| Body weight (kg) | 11.4 ± 0.3 | 14.7 ± 0.4* | 19.3 ± 1.7*† |
| Height (cm) | 43.3 ± 0.3 | 44.2 ± 0.8 | 45.3 ± 0.3 |
| Body length (cm) | 50.3 ± 0.6 | 50.3 ± 0.5 | 52.5 ± 0.3*† |
| BCS | 2.3 ± 0.3 | 5.5 ± 0.2* | 7.8 ± 0.3*† |
| Fat area (cm2) | 2.0 ± 0.5 | 17.0 ± 5.6 | 66.1 ± 14.7*† |
Significantly (P < 0.05) different from the value for this variable in the thin body condition.
Significantly (P < 0.05) different from the value for this variable in the optimal body condition.
BCS = Body condition score (9-point scale).
Serum leptin concentrations—Baseline serum leptin concentrations in the thin, optimal, and obese body conditions were 2.1 ± 0.5 ng/mL, 3.6 ± 0.6 ng/mL, and 5.4 ± 1.4 ng/mL, respectively. In the fed or unfed states in the thin body condition, serum leptin concentrations did not change throughout the experiment (Figure 1). In the fed state in the optimal body condition, there was a transient increase in serum leptin concentration (6.3 ± 1.0 ng/mL) at 4 PM and the concentrations returned to baseline value at 10 PM. In the fed state in the obese body condition, there was a significant increase in serum leptin concentration (12.3 ± 2.8 ng/mL) at 4 PM and the concentrations remained high at 10 PM. For the unfed states in the optimal and obese conditions, there was a gradual decrease in the serum leptin concentrations with time. There was good correlation between the serum leptin concentrations and fat area at the level of the L3 vertebra at 8 AM (n = 12; r = 0.938; P < 0.01), 4 PM (12; r = 0.961; P < 0.01), and 10 PM (12; r = 0.981; P < 0.01).
Mean ± SEM serum leptin concentrations in 4 dogs in thin (A), optimal (B), and obese body conditions (C) for each of 2 states (fed [black circles] and unfed states [white triangles]). In the fed state, dogs received food at 9 AM. *Value significantly (P < 0.05) different from the value at 8 AM. Value significantly (P < 0.05) different from values in the unfed state.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.2006
CSF leptin concentrations—The baseline CSF leptin concentrations in the thin, optimal, and obese body conditions were 24.3 ± 2.7 pg/mL, 86.1 ± 14.7 pg/mL, and 116.2 ± 47.3 pg/mL, respectively. In the fed state in the thin body condition, CSF leptin concentration was slightly increased (44.0 ± 11.5 pg/mL) from baseline value at 4 PM and returned to baseline value at 10 PM (Figure 2). In the optimal body condition, feeding maintained the CSF leptin concentration at the baseline value until 10 PM but the concentration gradually decreased in the unfed state. In the fed state in the obese body condition, CSF leptin concentration increased as the experiment progressed and a high concentration (313.3 ± 108.7 pg/mL) was detected at 10 PM. There was good correlation between CSF leptin concentration and fat area at the level of the L3 vertebra at 8 AM (n = 12; r = 0.917; P < 0.01), 4 PM (12; r = 0.935; P < 0.01), and 10 PM (12; r = 0.969; P < 0.01).
Mean ± SEM CSF leptin concentrations in 4 dogs in thin (A), optimal (B), and obese body conditions (C) for each of 2 states. In the fed state, dogs received food at 9 AM. See Figure 1 for key.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.2006
Leptin transport across the BBB—Efficacy of leptin transport across the BBB was evaluated by dividing the CSF leptin concentrations by the serum leptin concentrations to obtain the CSFL:SL ratio (Figure 3). There was no correlation between the CSF:SL ratio and fat area at the level of the L3 vertebra at 8 AM. A significant negative correlation (n = 12; r = −0.614; P < 0.05) between the CSF:SL ratio and fat area at the level of the L3 vertebra was evident at 4 PM, but the correlation was no longer significant at 10 PM.
Correlations between the CSFL:SL ratio (ie, CSF leptin concentrations divided by serum leptin concentrations) and fat area at the level of the third lumbar vertebra (determined via computed tomography) in 4 dogs in different body conditions at 8 AM (A), 4 PM (B), and 10 PM (C) during experiments in which food was provided at 9 AM. In each panel, the 12 datum points represent the values for each dog in the thin (black circles), optimal (white triangles), and obese body conditions (black squares). The line in panel B represents a regression line. NS = Not significant.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.2006
Discussion
To our knowledge, the present study is the first to report CSF leptin concentrations in dogs. There was a positive correlation between CSF leptin concentration and fat area at the level of the L3 vertebra in the 4 study dogs. This result was consistent with findings of previous studies24,25 in humans, which have indicated that CSF leptin concentrations are correlated with the body mass indices.
The negative correlation between the CSFL:SL ratio and body fat mass means that efficacy of leptin transport into the brain is decreased in obese individuals.15,26 In the present study, there was a significant negative correlation between the CSFL:SL ratio and fat area at the level of the L3 vertebra, suggesting that the dogs had acquired decreased efficacy of leptin transport into the brain during development of obesity. Thus, decreased efficacy of leptin transport at the BBB may also be one of the mechanisms of leptin resistance in dogs. Because leptin crosses the BBB via a saturable transport system,14 the transport ratio of blood leptin into the brain is decreased in animals with higher blood leptin concentrations.15,27 In addition, the decrease in leptin transport is not only attributable to the saturable transport system but is also the result of a transporter defect.17 It has been suggested that triglyceride-mediated leptin resistance is related to a leptin transporter defect.28 The decrease in efficacy of leptin transport in dogs could possibly be associated with similar mechanisms.
In our study, however, a significant correlation between the CSFL:SL ratio and fat area at the L3 level was evident only at 4 PM (7 hours after feeding) and was not evident at 8 AM (after food had been withheld for 24 hours and before feeding) or 10 PM (13 hours after feeding). This is an indication that there was no decrease in efficacy of leptin transport across the BBB before feeding. In mice, amelioration of leptin resistance at the BBB after withholding of food for 24 hours has been reported previously.29 Our data suggest that leptin transport into the brain is maintained during most of the day if dogs received food once a day. Thus, it seems that leptin resistance at the BBB is not an important cause of development of obesity in dogs.
The results of the present study were not consistent with those of previous studies15,26 in humans, which indicated that the CSFL:SL ratio before meals was correlated with body fat mass. The previous studies provided evidence to support the hypothesis that leptin resistance at the BBB is correlated with the cause of obesity in humans.30 In addition, unlike the results of the present study, no diurnal variation in leptin concentrations has been detected in the CSF of humans31 and rats.32 Although there may be a species difference, the inconsistencies probably reflect the difference in the fasting periods of these various studies. In most of these previous studies in humans, the patients fasted overnight and had 3 or 4 meals a day during the experiment. In the present study, food was withheld from the dogs for 24 hours before each experiment and they received food once in the morning thereafter. Because meals increase serum leptin concentrations,33,34 the patients who had several meals during an experiment might have maintained higher serum leptin concentrations throughout the day. As such, the higher serum leptin concentrations in the humans in those studies might have already saturated the transport of leptin into the brain because leptin transport at the BBB appears to be saturated at physiologic blood leptin concentration.31 This saturation of leptin transport may help to explain the differences in CSFL:SL ratio and diurnal variations in CSF leptin concentrations between the present and previous studies. Indeed, withholding of food for 18 hours decreased CSF leptin concentrations in normal and obese rats.35
As the other mechanism of leptin resistance, defects of leptin receptor-postreceptor signaling have been suspected.36–38 Leptin binds to its receptor in the hypothalamus to activate signaling, including the Janus activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway.39 In mice with diet-induced obesity, leptin (administered intracerebroventricularly) failed to activate STAT3 signaling, and this suggested that postreceptor signaling might play a role in leptin resistance.40 It has also been reported that the suppresser of cytokine signaling 3 (SOCS3) may have a correlation with leptin signaling and may be the cause of leptin resistance.36,37 Other mechanisms of leptin resistance in dogs remain to be determined.
In the present study, serum leptin concentrations increased 7 hours after feeding and returned to baseline value 13 hours after feeding in dogs in optimal body condition. The postprandial increase in serum leptin concentrations was consistent with data from previous studies22,34 in dogs. In the dogs in the obese body condition, high serum leptin concentrations were detected not only 7 hours after feeding but also 13 hours after feeding. This means that the obese dogs retained high serum leptin concentrations longer than dogs in the optimal body condition. Consistent with this finding, it is known that the period of fasting required to achieve a nadir serum leptin concentration is longer in obese humans than it is in lean humans.41 However, our data were different from those of previous studies42,43 in humans, which indicated that obese and lean subjects had similar circadian rhythms with regard to blood leptin concentrations. As discussed previously, humans in most studies have several meals and might maintain higher blood leptin concentrations during the experiments; this possibly minimized the differences in the postprandial variations of serum leptin concentration between obese and lean humans. In contrast, the present data have suggested that there was no postprandial variation in serum leptin concentration in the thin body condition in dogs. The attenuation of postprandial serum leptin response was consistent with the results obtained in the chronic nutritional deficiency model for athletic women.44 Hence, adaptations to chronic under-nutrition might be associated with attenuation of the increase in serum leptin in thin dogs.
Serum leptin concentrations have been reported as a good marker of obesity in dogs.45 In our study, there was a correlation between the serum leptin concentration and the fat area at the level of the L3 vertebra at all times after feeding, and this validated serum leptin concentration as a marker of the degree of obesity in dogs. However, depending on the degree of obesity, there was variation in the serum leptin response of the dogs to feeding. To avoid the effect of obesity on the variation of postprandial serum leptin response, blood samples should be collected at the same time after feeding so that the serum leptin concentrations can be used as a marker of the degree of obesity.
Without doubt, a limitation of the present study was the absence of data from female dogs. Because blood and CSF leptin concentrations are higher in women than men,42,46 the results obtained in this study may not hold true for female dogs. Further investigation is warranted to ascertain whether these data are applicable to dogs of either sex.
ABBREVIATIONS
| BBB | Blood-brain barrier |
| CSFL:SL ratio | CSF leptin concentration–to–serum leptin concentration ratio |
Science Diet Growth, Hill's Colgate, Tokyo, Japan.
Science Diet Maintenance, Hill's Colgate, Tokyo, Japan.
Domitor, Meiji Seika Kaisha, Tokyo, Japan.
Antisedan, Meiji Seika Kaisha, Tokyo, Japan.
Albumin bovine, Sigma Aldrich, Steinheim, Germany.
Amicon Ultra-4 5,000 MWCO, Millipore, Bedford, Mass.
Fuji Drychem system, Fujifilm Medical, Osaka, Japan.
References
- 1↑
Edney AT, Smith PM. Study of obesity in dogs visiting veterinary practices in the United Kingdom. Vet Rec 1986;118:391–396.
- 2
McGreevy PD, Thomson PC & Pride C, et al. Prevalence of obesity in dogs examined by Australian veterinary practices and the risk factors involved. Vet Rec 2005;156:695–702.
- 3↑
Mattheeuws D, Rottiers R & Kaneko JJ, et al. Diabetes mellitus in dogs: relationship of obesity to glucose tolerance and insulin response. Am J Vet Res 1984;45:98–103.
- 4↑
Lawler DF, Evans RH & Larson BT, et al. Influence of lifetime food restriction on causes, time, and predictors of death in dogs. J Am Vet Med Assoc 2005;226:225–231.
- 5↑
Butterwick RF, Hawthorne AJ. Advances in dietary management of obesity in dogs and cats. J Nutr 1998;128:2771S–2775S.
- 6↑
Zhang Y, Proenca R & Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–432.
- 8↑
Van Harmelen V, Reynisdottir S & Eriksson P, et al. Leptin secretion from subcutaneous and visceral adipose tissue in women. Diabetes 1998;47:913–917.
- 9↑
Frederich RC, Hamann A & Anderson S, et al. Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med 1995;1:1311–1314.
- 10
Salbe AD, Nicolson M, Ravussin E. Total energy expenditure and the level of physical activity correlate with plasma leptin concentrations in five-year-old children. J Clin Invest 1997;99:592–595.
- 11↑
Sagawa MM, Nakadomo F & Honjoh T, et al. Correlation between plasma leptin concentration and body fat content in dogs. Am J Vet Res 2002;63:7–10.
- 12↑
Considine RV, Caro JF. Leptin and the regulation of body weight. Int J Biochem Cell Biol 1997;29:1255–1272.
- 13
Malik KF, Young WS III. Localization of binding sites in the central nervous system for leptin (OB protein) in normal, obese (ob/ob), and diabetic (db/db) C57BL/6J mice. Endocrinology 1996;137:1497–1500.
- 14↑
Banks WA, Kastin AJ & Huang W, et al. Leptin enters the brain by a saturable system independent of insulin. Peptides 1996;17:305–311.
- 15↑
Caro JF, Kolaczynski JW & Nyce MR, et al. Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet 1996;348:159–161.
- 16
Van Heek M, Compton DS & France CF, et al. Diet-induced obese mice develop peripheral, but not central, resistance to leptin. J Clin Invest 1997;99:385–390.
- 17↑
Banks WA, DiPalma CR, Farrell CL. Impaired transport of leptin across the blood-brain barrier in obesity. Peptides 1999;20:1341–1345.
- 18
Halaas JL, Boozer C & Blair-West J, et al. Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci U S A 1997;94:8878–8883.
- 20↑
Ishioka K, Okumura M & Sagawa M, et al. Computed tomographic assessment of body fat in beagles. Vet Radiol Ultrasound 2005;46:49–53.
- 22↑
Nishii N, Takasu M & Ohba Y, et al. Effects of administration of glucocorticoids and feeding status on plasma leptin concentrations in dogs. Am J Vet Res 2006;67:266–270.
- 23↑
Iwase M, Kimura K & Komagome R, et al. Sandwich enzymelinked immunosorbent assay of canine leptin. J Vet Med Sci 2000;62:207–209.
- 24
Mantzoros C, Flier JS & Lesem MD, et al. Cerebrospinal fluid leptin in anorexia nervosa: correlation with nutritional status and potential role in resistance to weight gain. J Clin Endocrinol Metab 1997;82:1845–1851.
- 25
Dötsch J, Adelmann M & Englaro P, et al. Relation of leptin and neuropeptide Y in human blood and cerebrospinal fluid. J Neurol Sci 1997;151:185–188.
- 26
Schwartz MW, Peskind E & Raskind M, et al. Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. Nat Med 1996;2:589–593.
- 27
Krotkiewski M, Holmgren E & Karlsson U, et al. Weight loss and cerebrospinal-fluid leptin in obesity. Lancet 1998;351:415–416.
- 28↑
Banks WA, Coon AB & Robinson SM, et al. Triglycerides induce leptin resistance at the blood-brain barrier. Diabetes 2004;53:1253–1260.
- 29↑
Banks WA, Farrell CL. Impaired transport of leptin across the blood-brain barrier in obesity is acquired and reversible. Am J Physiol Endocrinol Metab 2003;285:E10–E15.
- 31↑
Wong ML, Licinio J & Yildiz BO, et al. Simultaneous and continuous 24-hour plasma and cerebrospinal fluid leptin measurements: dissociation of concentrations in central and peripheral compartments. J Clin Endocrinol Metab 2004;89:258–265.
- 32↑
Asakuma S, Hiraku O & Kurose Y, et al. Diurnal rhythm of cerebrospinal fluid and plasma leptin levels related to feeding in nonlactating and lactating rats. J Endocrinol 2004;180:283–286.
- 33
Schoeller DA, Cella LK & Sinha MK, et al. Entrainment of the diurnal rhythm of plasma leptin to meal timing. J Clin Invest 1997;100:1882–1887.
- 34
Ishioka K, Hatai H & Komabayashi K, et al. Diurnal variations of serum leptin in dogs: effects of fasting and re-feeding. Vet J 2005;169:85–90.
- 35↑
O'Doherty RM, Nguyen L. Blunted fasting-induced decreases in plasma and CSF leptin concentrations in obese rats: the role of increased leptin secretion. Int J Obes Relat Metab Disord 2004;28:173–175.
- 36
Bjørbaek C, Elmquist JK & Frantz JD, et al. Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell 1998;1:619–625.
- 37
Mori H, Hanada R & Hanada T, et al. Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity. Nat Med 2004;10:739–743.
- 38
Münzberg H, Flier JS, Bjørbaek C. Region-specific leptin resistance within the hypothalamus of diet-induced obese mice. Endocrinology 2004;145:4880–4889.
- 39↑
Sahu A. Minireview: a hypothalamic role in energy balance with special emphasis on leptin. Endocrinology 2004;145:2613–2620.
- 40↑
El-Haschimi K, Pierroz DD & Hileman SM, et al. Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest 2000;105:1827–1832.
- 41↑
Boden G, Chen X & Mozzoli M, et al. Effect of fasting on serum leptin in normal human subjects. J Clin Endocrinol Metab 1996;81:3419–3423.
- 42
Saad MF, Riad-Gabriel MG & Khan A, et al. Diurnal and ultradian rhythmicity of plasma leptin: effects of gender and adiposity. J Clin Endocrinol Metab 1998;83:453–459.
- 43
Langendonk JG, Pijl H & Toornvliet AC, et al. Circadian rhythm of plasma leptin levels in upper and lower body obese women: influence of body fat distribution and weight loss. J Clin Endocrinol Metab 1998;83:1706–1712.
- 44↑
Laughlin GA, Yen SS. Hypoleptinemia in women athletes: absence of a diurnal rhythm with amenorrhea. J Clin Endocrinol Metab 1997;82:318–321.
- 45↑
Ishioka K, Soliman MM & Sagawa M, et al. Experimental and clinical studies on plasma leptin in obese dogs. J Vet Med Sci 2002;64:349–353.
- 46
Landt M, Parvin CA, Wong M. Leptin in cerebrospinal fluid from children: correlation with plasma leptin, sexual dimorphism, and lack of protein binding. Clin Chem 2000;46:854–858.
