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Short-term effects of energy changes on plasma leptin concentrations and glucose tolerance in healthy ponies
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The Veterinary Journal The Veterinary Journal 178 (2008) 233–237 www.elsevier.com/locate/tvjl
Short-term effects of energy changes on plasma leptin concentrations and glucose tolerance in healthy ponies Stephanie Van Weyenberg a,*, Myriam Hesta a, Johan Buyse b, Georgios A. Papadopoulos a, Geert P.J. Janssens a b
a Laboratory of Animal Nutrition, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium Laboratory of Physiology, Immunology and Genetics of Domestic Animals, Catholic University of Leuven, Belgium
Accepted 4 July 2007
Abstract To determine whether plasma leptin concentrations and glucose tolerance are affected by changes in energy balance, nine healthy Shetland ponies were fed at 140% followed by 75% of their maintenance requirements for 13 days in each of the two periods. Bodyweight was recorded every three days. Blood samples were taken every two days and analysed for leptin and cortisol. An oral glucose tolerance test was performed on day 7 of each period. Serial blood samples were analysed for glucose and insulin. Although bodyweight was not affected, plasma leptin concentrations increased (P < 0.001) initially during overfeeding, but returned to previous values after 7 days. During underfeeding, plasma leptin concentrations decreased (P < 0.001). Underfeeding was associated with a higher AUC for plasma glucose (P = 0.02) and plasma insulin (P = 0.05) resulting in a decreased glucose tolerance (AUC glucose/AUC insulin; P = 0.008), probably due to a plasma cortisol increase caused by the reduced feed intake. It is concluded that changes in energy balance, without altering bodyweight, can influence plasma leptin concentrations in ponies. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Leptin; Glucose tolerance; Energy balance; Equine
Introduction Obesity in equines was associated with high plasma leptin concentrations (Gentry et al., 2002) and an increased insulin response to glucose (Jeffcott et al., 1986; Hoffman et al., 2003), suggesting that leptin concentrations can be used to predict body fat mass (Kearns et al., 2006) and insulin resistance (IR). However, studies in horses have shown a wide range of leptin concentrations in animals with a similar degree of obesity (Gentry and Thompson, 2002), so clearly other factors in addition to adipose mass modulate leptin secretion.
*
Corresponding author. Tel.: + 32 9 264 78 28; fax: + 32 9 264 78 48. E-mail address: [email protected] (S.V. Weyenberg). 1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.07.018
The state of energy balance may affect plasma leptin concentration. Decreases due to short-term fasting do not correspond to the amount of fat mass lost in horses (Buff et al., 2005). This is also the case in humans where changes in leptin concentrations have been shown to be due to low energy intake for 7 days (Dubuc et al., 1998) or short-term massive overfeeding for 12 h (Kolaczynski et al., 1996). Moreover, obese horses with high plasma leptin concentrations have higher plasma insulin and triiodothyronine (T3) concentrations as well as decreased insulin sensitivity compared to similarly obese horses with low plasma leptin concentrations (Cartmill et al., 2003). This might suggest that high concentrations of leptin rather than obesity alone might be responsible for the development of IR, as has been reported in humans (Taylor et al., 1996). The present study tested the hypothesis that plasma leptin concentrations are affected by a sequence of short-term
234
S.V. Weyenberg et al. / The Veterinary Journal 178 (2008) 233–237
over- and underfeeding in ponies with the same body condition score, and that energy balance should be taken into account when plasma leptin concentrations are used as predictor for fat mass and IR. Materials and methods The experimental design was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University. Nine healthy Shetland ponies with an average age of 6 years (±2 years) were regularly dewormed, vaccinated and dentally assessed. The ponies were placed in individual stables (8 m2) on rubber mats. The lighting schedule provided 15 h lighting per day. Initial bodyweight (BW) ranged from 156 kg (±28 kg) with an initial body condition score (BCS) of 4 or 5 (1 = cachexia; 5 = normal; 9 = highly obese), according to the ranking proposed by Henneke et al. (1983). A mixture of alfalfa, hay and straw (5.7% moisture, 12% crude protein (CP), 4.6% ether extract (EE), 31% crude fibre (CF), 12% crude ash (Ash) and 41% nitrogen free extract (NFE)) was supplemented with a commercial concentrate (Essential, Cavalor) (6.9% moisture, 13% CP, 4.1% EE, 8.93% CF, 7.8% Ash and 66% NFE) and offered twice daily (09:00 and 18:00 h) according to CVB-recommendations for energy supply (Centraal Veevoederbureau, 1996). On average, the total daily amount of feed provided was 2.37 kg (±0.32 kg) corresponding with 347 kJ NE/ kg0.75 BW. The roughage:concentrate ratio was 1:1 on a fresh matter basis. The ponies had unlimited access to water during the entire experiment. After three weeks’ adaptation, during which time no BW change was recorded, feed intake increased to 140% of maintenance energy requirements, corresponding to 486 kJ NE/kg0.75 BW. Total average daily amount of feed provided was 1.08 (±0.15 kg) and 2.00 kg (±0.27 kg) for roughage and concentrates, respectively. After 13 days, feed intake was restricted to 0.88 kg (±0.12 kg) roughage and 0.82 kg (±0.11 kg) concentrates or 75% of the initial maintenance requirements (260 kJ NE/kg0.75 BW). During the entire trial, all ponies completed their meals within 1 h. The high roughage:concentrate ratio (1:2) during the first period was chosen to reduce intake time. Higher proportions of roughage, with longer intake times, especially in the evening, would reduce the period of overnight feed deprivation, which could influence plasma leptin concentrations in the morning (McManus and Fitzgerald, 2000). Although total CF concentration was still 15.32% on a dry matter (DM) basis and only slightly lower than the recommendations, the proportion of roughage was increased during the underfeeding period to prevent gastrointestinal problems. Due to the different roughage:concentrate ratios during overand underfeeding, 1:2 and 1:1, respectively, the nutrient compositions of the two complete diets were not equal, but still moderate (Table 1) and not thought to overrule the energy balance effects. A cross-over design was not used because a change in energy balance cannot be separated from the preceding nutritional status. For the same reason, a period in-between studies, in which the animals would have been fed at 100% of maintenance requirements, was not included as this would have meant a change in energy balance.
Table 1 Proximate analysis of the two complete diets (g/kg fresh matter) Period
1
2
Energy intake as % of maintenance requirement
140%
75%
Moisture Crude ash Ether extract Crude protein Crude fibre Nitrogen free extract Starch and sugar Calculated net energy (MJ/kg)
64.3 89.8 42.0 125 163 571 183 8.28
62.8 97.3 43.2 124 201 534 157 7.55
Bodyweight loss or gain was recorded every two days. Body condition score was determined at the end of each period. The short duration of the study was chosen not only to prevent changes in BW and BCS, but also to limit the effects of other external variables (such as season, daylight and temperature). To determine metabolic profile, morning blood samples (13 h of feed deprivation) were taken every 2 days between 08:00 and 09:00 h. After 7 days of over- and underfeeding (days 7 and 20) an oral glucose tolerance test (OGTT) was performed. Both OGTTs were performed under the same conditions as the daily blood sampling. After overnight feed deprivation, a jugular vein catheter was inserted between 06:00 and 07:00 h. Morning blood samples (13 h of feed deprivation) were taken between 08:30 and 09:30 h, at least 2 h after placing the catheters. Immediately after blood sampling, 1 g glucose/kg bodyweight of a 20% glucose solution was given by naso-oesophageal tube. Serial blood samples were taken at 0, 15, 30, 45, 60, 90, 120, 180, 240, 300 min after the glucose load for plasma glucose (Li–heparin tubes) and plasma insulin (Na-EDTA tubes) analyses. Tubes were centrifuged for 15 min at 1500g. Plasma samples were analysed for triglycerides (TG), non-esterified free fatty acids (NEFA), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), triiodothyroxine (T3), thyroxin (T4), leptin and cortisol concentrations (Li-heparin tubes). Plasma glucose and TG concentrations were measured spectrophotometrically (Monarch Chemistry System, B-1930). Plasma NEFA concentrations were measured by the WAKO NEFA C test kit (Wako Chemicals), modified for use in the Monarch Chemistry System. The plasma T3 and T4 levels were determined by using a specific radioimmunoassay as described by Darras et al. (1992). The intra-assay coefficients of variation (CV) were 4.1% and 4.7% for T3 and T4, respectively. Plasma insulin concentrations were assessed by radioimmunoassay (RIA). Plasma cortisol concentrations were also determined by RIA using a commercial 125I kit from ICN Biomedicals, demonstrating an intra-assay CV of 6.7%. All samples were run in the same assay in order to avoid intra-assay variability. Areas under the curve (AUC) of glucose and insulin were calculated as the sum of all trapezium surfaces. Leptin was measured using a multispecies RIA kit (Linco Research). The antibody used was guineapig anti-human leptin. In the absence of a purified equine leptin preparation, results are reported as human equivalents of immunoreactive leptin (ir-leptin). The intra-assay CV was 5.2%. Data are reported as means ± SD unless otherwise stated. Statistical analysis was performed with SPSS 14.0 (SPSS Inc.). Comparison between the treatments days was done by repeated measures analysis. Where appropriate, paired t-tests were performed. Because of non-normal distribution of data, AUC glucose and AUC insulin were analysed by means of the non-parametric Wilcoxon rank test. Statistical significance was considered when P < 0.05.
Results Throughout the whole of the experiment BCS and BW did not significantly change (Fig. 1). Plasma parameters are summarised in Table 2. Underfeeding resulted in an increased plasma NEFA (P < 0.001) concentration, while no changes due to treatments were seen for plasma TG (P = 0.243). The ponies responded to underfeeding by lower plasma CPK activity (P = 0.001). In contrast, plasma LDH activity was not significantly (P = 0.313) affected by caloric intake. Similarly, the plasma thyroid hormone concentrations, T3 (P = 0.607) and T4 (P = 0.318) were not influenced by short-term changes in energy balance. During the first five days of overfeeding, plasma leptin concentrations increased (P < 0.001) (Fig. 2). Although overfeeding continued for 13 days, plasma leptin concentrations returned to the initial values after seven days. Dur-
S.V. Weyenberg et al. / The Veterinary Journal 178 (2008) 233–237
235
40
200
a
Cortisol (mg/dL)
Bodyweight (kg)
35 150
100
50
30
b
25
b b
20
b bc
b
b b
b bc
c c c
15 10 5 0 0
0 0
4
8
12
16
20
24
4
8
28
12
16
20
24
28
Time (Days)
Time (days)
Table 2 Blood samples (means ± SD) taken between 08:00 and 09:00 h from nine ponies fed twice a day (09:00 and 18:00 h) at 140% and 75% of their requirements Period
1
2
Pvalue
Energy intake as % of maintenance requirement
140%
75%
NEFA (mmol/L) TG (mg/dL) LDH (IU/L) CPK (IU/L) T3 (ng/mL) T4 (ng/mL)
0.04 ± 0.01 18.5 ± 2.3 482 ± 23.9 182 ± 13.6 0.26 ± 0.02 19.2 ± 2.6
0.24 ± 0.07 19.0 ± 1.7 470 ± 20.8 151 ± 20.2 0.24 ± 0.04 20.3 ± 1.7
0.001 0.243 0.313 0.001 0.307 0.318
NEFA, non-esterified free fatty acids; TG, triglycerides; LDH, lactate dehydrogenase; CPK, creatine phosphokinase; T3, triiodothyroxine; T4, thyroxin.
Fig. 3. Plasma cortisol concentrations (mg/dL) in nine ponies fed twice a day (09:00 and 18:00 h) at 140% (closed diamonds) and 75% (open triangles) of their requirements. The oral glucose tests were performed on days 7 and 20 (indicated by the arrows). Data are summarised as means ± SE and significant difference (P < 0.05) are indicated by different letters.
Glucose (mg/dL)
Fig. 1. Bodyweight (means ± SE) in kg in nine ponies fed twice a day (09:00 and 18:00 h) at 140% (closed diamonds) and 75% (open triangles) of their requirements.
160 * *
120 *
*
80 40 0 0
60
120
180
240
300
Time after glucose load (min) Fig. 4. Mean plasma glucose concentration (mg/dL) of nine ponies after an oral glucose tolerance test performed after 7 days overfeeding (closed diamonds) and underfeeding (open triangles). Data are summarised as means ± SE and significant difference between feeding regimes at a similar time point are indicated with * for P < 0.05.
Ir Leptin (ng/mL)
4 ab
3
b
a b
b
2
b bc
c d d
d
d
cd
d
1 0 0
4
8
12
16
20
24
28
Time (Days) Fig. 2. Plasma leptin concentrations (ng/mL) in nine ponies fed twice a day (09:00 and 18:00 h) at 140% (closed diamonds) and 75% (open triangles) of their requirements. Data are summarised as means ± SE and significant difference (P < 0.05) are indicated by different letters.
ing feed restriction, plasma leptin concentration significantly decreased (P < 0.001). In general, plasma cortisol concentrations were higher during the underfeeding than during the overfeeding period, 26.0 ± 9.7 and 17.1 ± 4.7 mg/dL, respectively (P = 0.006) (Fig. 3). However, during the overfeeding period,
plasma cortisol concentrations were also elevated on days 7, 9 and 11, compared with days 1, 3 and 5 (P < 0.05). On day 20, when the oral glucose test during underfeeding took place, plasma cortisol levels of all ponies were extremely elevated (P < 0.001) and significantly higher (P = 0.009) compared to day 7 (the oral glucose test during overfeeding). During the OGTT, underfeeding was associated with a higher glucose concentration 180, 240 and 300 min after glucose administration (P < 0.05) (Fig. 4). AUC for glucose (P = 0.02) was significantly affected by treatment (P = 0.02) (29 ± 5 and 34 ± 7 g min dL-1 for over- and underfeeding, respectively). There was a trend towards a higher AUC for insulin during underfeeding (P = 0.05) (27 ± 16 and 45 ± 29 U min L 1 for over- and underfeeding, respectively). Discussion Bodyweight was not affected in any of the ponies so the results reported in this study were solely caused by changes
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in energy balance itself. Increased plasma NEFA concentrations during feed restriction are likely to be the result of an increased fatty acid mobilisation. However, parameters of metabolic rate, plasma T3 and T4 were not significantly changed, probably due to the short period of underfeeding (3 weeks) and the modest change in energy intake (75% of maintenance). Overfeeding in ponies resulted in a significant increase in leptin without changes in fat mass, as seen in humans (Kolaczynski et al., 1996). Most remarkable was that leptin concentrations returned to initial values within 7 days despite constant overfeeding. Changes in leptin may be explained by the lipostatic theory of bodyweight maintenance (Kennedy, 1953), which suggests that animals are able to maintain their bodyweight by adapting food intake and energy expenditure to available food (Buff et al., 2006) and energy requirements (Gordon et al., 2007). Besides reporting the status of body energy stores to the brain, leptin also functions as a sensor for energy balance. Hence, leptin is believed to play a key role in the lipostatic theory. In response to a positive energy balance, leptin will both reduce feed intake and increase metabolic rate in order to prevent changes in bodyweight. Additionally, leptin prevents further expression of the leptin gene due to a negative feedback mechanism and returns to initial concentrations (Houseknecht et al., 1998). In our study, underfeeding resulted in a decreased leptin concentration. Buff et al. (2006) also described a decrease in leptin in obese ponies receiving 75% of their ad libitum intake, and to a larger extent than in the present study. Placing the catheters before the OGTT resulted in elevated plasma cortisol concentrations (P < 0.05). However, plasma cortisol concentrations were higher during the second OGTT (P = 0.009), when ponies were in a negative energy balance (underfeeding). The effect of energy restriction on cortisol concentrations in literature is not clear. In contrast to the decreased cortisol concentration reported after prolonged nutrient restriction (Glade et al., 1984; Sticker et al., 1995), increased cortisol concentrations were found with hypoglycemia and acute feed restriction in horses (James et al., 1970) and donkeys (Forhead and Dobson, 1997). A clear interaction between energy status and plasma cortisol was therefore seen in our study. Catheter placement led to a higher cortisol increase during underfeeding than during the period of overfeeding. Considering that no elevated plasma cortisol concentrations were seen after consecutive OGTTs in other experiments using these same ponies (Van Weyenberg et al., unpublished data), it can be postulated that feed restriction was the main cause of the cortisol increase. During the OGTT, underfeeding was associated with a higher AUC for glucose (P = 0.02) and a trend for a higher AUC for insulin (P = 0.05). Consequently the higher glucose response during a negative energy balance was not the result of a decreased insulin secretion, but of a decreased glucose tolerance (AUC glucose/AUC insulin; P = 0.008). Diets higher in starch and sugar and lower in fibre, as fed
during overfeeding in this study, have been associated with insulin resistance (Hoffman et al., 2003). Thus, decreased glucose tolerance, seen during underfeeding was not caused by nutrient differences between the two periods and this higher glucose response could be explained by higher cortisol concentrations (Ralston, 2002). Conclusions Differences in feed intake and energy balance, separate from bodyweight changes, can influence plasma leptin concentrations and glucose tolerance in ponies. In addition, plasma leptin concentrations also changed as a function of the duration of the negative energy balance. For this reason both energy balance and the time after changed nutritional intake must be taken into account when leptin is used as predictor for fat mass or IR. Acknowledgements We thank Steven Galle for his excellent care of the ponies, Dirk Adriaensen for his help during the blood sampling and Herman De Rycke and Inge Vaesen for the analyses. References Buff, P.R., Johnson, P.J., Wiedmeyer, C.E., Ganjam, V.K., Messer, N.T., Keisler, D.H., 2006. Modulation of leptin, insulin, and growth hormone in obese pony mares under chronic nutritional restriction and supplementation with ractopamine hydrochloride. Veterinary Therapeutics 7, 64–72. Buff, P.R., Morrison, C.D., Ganjam, V.K., Keisler, D.H., 2005. Effects of short-term feed deprivation and melatonin implants on circadian patterns of leptin in the horse. Journal of Animal Science 83, 1023– 1032. Cartmill, J.A., Thompson, D.L., Storer, W.A., Gentry, L.R., Huff, N.K., 2003. Endocrine responses in mares and geldings with high body condition scores grouped by high vs low resting leptin concentrations. Journal of Animal Science 81, 2311–2321. Centraal Veevoederbureau, 1996. Het definitieve VEP- en VREp-systeem. CVB-rapport 15, pp 39–50. Darras, V.M., Visser, T.J., Berghman, L.R., Kuhn, E.R., 1992. Ontogeny of Type-I and Type-Iii deiodinase activities in embryonic and posthatch chicks – relationship with changes in plasma triiodothyronine and growth-hormone levels. Comparative Biochemistry and Physiology A-Physiology 103, 131–136. Dubuc, G.R., Phinney, S.D., Stern, J.S., Havel, P.J., 1998. Changes of serum leptin and endocrine and metabolic parameters after 7 days of energy restriction in men and women. Metabolism-Clinical and Experimental 47, 429–434. Forhead, A.J., Dobson, H., 1997. Plasma glucose and cortisol responses to exogenous insulin in fasted donkeys. Research in Veterinary Science 62, 265–269. Gentry, L.R., Thompson, D.L., 2002. The relationship between body condition, leptin, and reproductive and hormonal characteristics of mares during the seasonal anovulatory period. Theriogenology 58, 3– 566. Gentry, L.R., Thompson, D.L., Gentry, G.T., Davis, K.A., Godke, R.A., 2002. High versus low body condition in mares: Interactions with responses to somatotropin, GnRH analog, and dexamethasone. Journal of Animal Science 80, 3277–3285.
S.V. Weyenberg et al. / The Veterinary Journal 178 (2008) 233–237 Glade, M.J., Gupta, S., Reimers, T.J., 1984. Hormonal responses to high and low planes of nutrition in weanling thoroughbreds. Journal of Animal Science 59, 658–665. Gordon, M.E., McKeever, K.H., Betros, C.L., Manso, H.C., 2007. Plasma leptin, ghrelin and adiponectin concentrations in young fit racehorses versus mature unfit Standardbreds. The Veterinary Journal 173, 91–100. Henneke, D.R., Potter, G.D., Kreider, J.L., Yeates, B.F., 1983. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Veterinary Journal 15, 371–372. Hoffman, R.M., Boston, R.C., Stefanovski, D., Kronfeld, D.S., Harris, P.A., 2003. Obesity and diet affect glucose dynamics and insulin sensitivity in Thoroughbred geldings. Journal of Animal Science 81, 2333–2342. Houseknecht, K.L., Baile, C.A., Matteri, R.L., Spurlock, M.E., 1998. The biology of leptin: A review. Journal of Animal Science 76, 1405–1420. James, V.H.T., Horner, M.W., Moss, M.S., Rippon, A.E., 1970. Adrenocortical function in the horse. Journal of Endocrinology 48, 319– 335. Jeffcott, L.B., Field, J.R., Mclean, J.G., Odea, K., 1986. Glucose-tolerance and insulin sensitivity in ponies and standard-bred horses. Equine Veterinary Journal 18, 97–101.
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Kearns, C.F., McKeever, K.H., Roegner, V., Brady, S.M., Malinowski, K., 2006. Adiponectin and leptin are related to fat mass in horses. The Veterinary Journal 172, 460–465. Kennedy, G.C., 1953. The role of depot fat in the hypothalamic control of food intake in the rat. Proceedings of the Royal Society of London series B-Biological Science 140, 578–596. Kolaczynski, J.W., Ohannesian, J.P., Considine, R.V., Marco, C.C., Caro, J.F., 1996. Response of leptin to short-term and prolonged overfeeding in humans. Journal of Clinical Endocrinology and Metabolism 81, 4162–4165. McManus, C.J., Fitzgerald, B.P., 2000. Effects of a single day of feed restriction on changes in serum leptin, gonadotropins, prolactin, and metabolites in aged and young mares. Domestic Animal Endocrinology 19, 1–13. Ralston, S.L., 2002. Insulin and glucose regulation. Veterinary Clinics of North America-Equine Practice 18, 295–304. Sticker, L.S., Thompson, D.L., Fernandez, J.M., Bunting, L.D., Depew, C.L., 1995. Dietary-protein and (or) energy restriction in mares – plasma growth-hormone, Igf-I, prolactin, cortisol, and thyroid-hormone responses to feeding, glucose, and epinephrine. Journal of Animal Science 73, 1424–1432. Taylor, S.I., Barr, V., Reitman, M., 1996. Medicine – does leptin contribute to diabetes caused by obesity? Science 274, 1151–1152.
The Veterinary Journal The Veterinary Journal 178 (2008) 233–237 www.elsevier.com/locate/tvjl
Short-term effects of energy changes on plasma leptin concentrations and glucose tolerance in healthy ponies Stephanie Van Weyenberg a,*, Myriam Hesta a, Johan Buyse b, Georgios A. Papadopoulos a, Geert P.J. Janssens a b
a Laboratory of Animal Nutrition, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium Laboratory of Physiology, Immunology and Genetics of Domestic Animals, Catholic University of Leuven, Belgium
Accepted 4 July 2007
Abstract To determine whether plasma leptin concentrations and glucose tolerance are affected by changes in energy balance, nine healthy Shetland ponies were fed at 140% followed by 75% of their maintenance requirements for 13 days in each of the two periods. Bodyweight was recorded every three days. Blood samples were taken every two days and analysed for leptin and cortisol. An oral glucose tolerance test was performed on day 7 of each period. Serial blood samples were analysed for glucose and insulin. Although bodyweight was not affected, plasma leptin concentrations increased (P < 0.001) initially during overfeeding, but returned to previous values after 7 days. During underfeeding, plasma leptin concentrations decreased (P < 0.001). Underfeeding was associated with a higher AUC for plasma glucose (P = 0.02) and plasma insulin (P = 0.05) resulting in a decreased glucose tolerance (AUC glucose/AUC insulin; P = 0.008), probably due to a plasma cortisol increase caused by the reduced feed intake. It is concluded that changes in energy balance, without altering bodyweight, can influence plasma leptin concentrations in ponies. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Leptin; Glucose tolerance; Energy balance; Equine
Introduction Obesity in equines was associated with high plasma leptin concentrations (Gentry et al., 2002) and an increased insulin response to glucose (Jeffcott et al., 1986; Hoffman et al., 2003), suggesting that leptin concentrations can be used to predict body fat mass (Kearns et al., 2006) and insulin resistance (IR). However, studies in horses have shown a wide range of leptin concentrations in animals with a similar degree of obesity (Gentry and Thompson, 2002), so clearly other factors in addition to adipose mass modulate leptin secretion.
*
Corresponding author. Tel.: + 32 9 264 78 28; fax: + 32 9 264 78 48. E-mail address: [email protected] (S.V. Weyenberg). 1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2007.07.018
The state of energy balance may affect plasma leptin concentration. Decreases due to short-term fasting do not correspond to the amount of fat mass lost in horses (Buff et al., 2005). This is also the case in humans where changes in leptin concentrations have been shown to be due to low energy intake for 7 days (Dubuc et al., 1998) or short-term massive overfeeding for 12 h (Kolaczynski et al., 1996). Moreover, obese horses with high plasma leptin concentrations have higher plasma insulin and triiodothyronine (T3) concentrations as well as decreased insulin sensitivity compared to similarly obese horses with low plasma leptin concentrations (Cartmill et al., 2003). This might suggest that high concentrations of leptin rather than obesity alone might be responsible for the development of IR, as has been reported in humans (Taylor et al., 1996). The present study tested the hypothesis that plasma leptin concentrations are affected by a sequence of short-term
234
S.V. Weyenberg et al. / The Veterinary Journal 178 (2008) 233–237
over- and underfeeding in ponies with the same body condition score, and that energy balance should be taken into account when plasma leptin concentrations are used as predictor for fat mass and IR. Materials and methods The experimental design was approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University. Nine healthy Shetland ponies with an average age of 6 years (±2 years) were regularly dewormed, vaccinated and dentally assessed. The ponies were placed in individual stables (8 m2) on rubber mats. The lighting schedule provided 15 h lighting per day. Initial bodyweight (BW) ranged from 156 kg (±28 kg) with an initial body condition score (BCS) of 4 or 5 (1 = cachexia; 5 = normal; 9 = highly obese), according to the ranking proposed by Henneke et al. (1983). A mixture of alfalfa, hay and straw (5.7% moisture, 12% crude protein (CP), 4.6% ether extract (EE), 31% crude fibre (CF), 12% crude ash (Ash) and 41% nitrogen free extract (NFE)) was supplemented with a commercial concentrate (Essential, Cavalor) (6.9% moisture, 13% CP, 4.1% EE, 8.93% CF, 7.8% Ash and 66% NFE) and offered twice daily (09:00 and 18:00 h) according to CVB-recommendations for energy supply (Centraal Veevoederbureau, 1996). On average, the total daily amount of feed provided was 2.37 kg (±0.32 kg) corresponding with 347 kJ NE/ kg0.75 BW. The roughage:concentrate ratio was 1:1 on a fresh matter basis. The ponies had unlimited access to water during the entire experiment. After three weeks’ adaptation, during which time no BW change was recorded, feed intake increased to 140% of maintenance energy requirements, corresponding to 486 kJ NE/kg0.75 BW. Total average daily amount of feed provided was 1.08 (±0.15 kg) and 2.00 kg (±0.27 kg) for roughage and concentrates, respectively. After 13 days, feed intake was restricted to 0.88 kg (±0.12 kg) roughage and 0.82 kg (±0.11 kg) concentrates or 75% of the initial maintenance requirements (260 kJ NE/kg0.75 BW). During the entire trial, all ponies completed their meals within 1 h. The high roughage:concentrate ratio (1:2) during the first period was chosen to reduce intake time. Higher proportions of roughage, with longer intake times, especially in the evening, would reduce the period of overnight feed deprivation, which could influence plasma leptin concentrations in the morning (McManus and Fitzgerald, 2000). Although total CF concentration was still 15.32% on a dry matter (DM) basis and only slightly lower than the recommendations, the proportion of roughage was increased during the underfeeding period to prevent gastrointestinal problems. Due to the different roughage:concentrate ratios during overand underfeeding, 1:2 and 1:1, respectively, the nutrient compositions of the two complete diets were not equal, but still moderate (Table 1) and not thought to overrule the energy balance effects. A cross-over design was not used because a change in energy balance cannot be separated from the preceding nutritional status. For the same reason, a period in-between studies, in which the animals would have been fed at 100% of maintenance requirements, was not included as this would have meant a change in energy balance.
Table 1 Proximate analysis of the two complete diets (g/kg fresh matter) Period
1
2
Energy intake as % of maintenance requirement
140%
75%
Moisture Crude ash Ether extract Crude protein Crude fibre Nitrogen free extract Starch and sugar Calculated net energy (MJ/kg)
64.3 89.8 42.0 125 163 571 183 8.28
62.8 97.3 43.2 124 201 534 157 7.55
Bodyweight loss or gain was recorded every two days. Body condition score was determined at the end of each period. The short duration of the study was chosen not only to prevent changes in BW and BCS, but also to limit the effects of other external variables (such as season, daylight and temperature). To determine metabolic profile, morning blood samples (13 h of feed deprivation) were taken every 2 days between 08:00 and 09:00 h. After 7 days of over- and underfeeding (days 7 and 20) an oral glucose tolerance test (OGTT) was performed. Both OGTTs were performed under the same conditions as the daily blood sampling. After overnight feed deprivation, a jugular vein catheter was inserted between 06:00 and 07:00 h. Morning blood samples (13 h of feed deprivation) were taken between 08:30 and 09:30 h, at least 2 h after placing the catheters. Immediately after blood sampling, 1 g glucose/kg bodyweight of a 20% glucose solution was given by naso-oesophageal tube. Serial blood samples were taken at 0, 15, 30, 45, 60, 90, 120, 180, 240, 300 min after the glucose load for plasma glucose (Li–heparin tubes) and plasma insulin (Na-EDTA tubes) analyses. Tubes were centrifuged for 15 min at 1500g. Plasma samples were analysed for triglycerides (TG), non-esterified free fatty acids (NEFA), lactate dehydrogenase (LDH), creatine phosphokinase (CPK), triiodothyroxine (T3), thyroxin (T4), leptin and cortisol concentrations (Li-heparin tubes). Plasma glucose and TG concentrations were measured spectrophotometrically (Monarch Chemistry System, B-1930). Plasma NEFA concentrations were measured by the WAKO NEFA C test kit (Wako Chemicals), modified for use in the Monarch Chemistry System. The plasma T3 and T4 levels were determined by using a specific radioimmunoassay as described by Darras et al. (1992). The intra-assay coefficients of variation (CV) were 4.1% and 4.7% for T3 and T4, respectively. Plasma insulin concentrations were assessed by radioimmunoassay (RIA). Plasma cortisol concentrations were also determined by RIA using a commercial 125I kit from ICN Biomedicals, demonstrating an intra-assay CV of 6.7%. All samples were run in the same assay in order to avoid intra-assay variability. Areas under the curve (AUC) of glucose and insulin were calculated as the sum of all trapezium surfaces. Leptin was measured using a multispecies RIA kit (Linco Research). The antibody used was guineapig anti-human leptin. In the absence of a purified equine leptin preparation, results are reported as human equivalents of immunoreactive leptin (ir-leptin). The intra-assay CV was 5.2%. Data are reported as means ± SD unless otherwise stated. Statistical analysis was performed with SPSS 14.0 (SPSS Inc.). Comparison between the treatments days was done by repeated measures analysis. Where appropriate, paired t-tests were performed. Because of non-normal distribution of data, AUC glucose and AUC insulin were analysed by means of the non-parametric Wilcoxon rank test. Statistical significance was considered when P < 0.05.
Results Throughout the whole of the experiment BCS and BW did not significantly change (Fig. 1). Plasma parameters are summarised in Table 2. Underfeeding resulted in an increased plasma NEFA (P < 0.001) concentration, while no changes due to treatments were seen for plasma TG (P = 0.243). The ponies responded to underfeeding by lower plasma CPK activity (P = 0.001). In contrast, plasma LDH activity was not significantly (P = 0.313) affected by caloric intake. Similarly, the plasma thyroid hormone concentrations, T3 (P = 0.607) and T4 (P = 0.318) were not influenced by short-term changes in energy balance. During the first five days of overfeeding, plasma leptin concentrations increased (P < 0.001) (Fig. 2). Although overfeeding continued for 13 days, plasma leptin concentrations returned to the initial values after seven days. Dur-
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235
40
200
a
Cortisol (mg/dL)
Bodyweight (kg)
35 150
100
50
30
b
25
b b
20
b bc
b
b b
b bc
c c c
15 10 5 0 0
0 0
4
8
12
16
20
24
4
8
28
12
16
20
24
28
Time (Days)
Time (days)
Table 2 Blood samples (means ± SD) taken between 08:00 and 09:00 h from nine ponies fed twice a day (09:00 and 18:00 h) at 140% and 75% of their requirements Period
1
2
Pvalue
Energy intake as % of maintenance requirement
140%
75%
NEFA (mmol/L) TG (mg/dL) LDH (IU/L) CPK (IU/L) T3 (ng/mL) T4 (ng/mL)
0.04 ± 0.01 18.5 ± 2.3 482 ± 23.9 182 ± 13.6 0.26 ± 0.02 19.2 ± 2.6
0.24 ± 0.07 19.0 ± 1.7 470 ± 20.8 151 ± 20.2 0.24 ± 0.04 20.3 ± 1.7
0.001 0.243 0.313 0.001 0.307 0.318
NEFA, non-esterified free fatty acids; TG, triglycerides; LDH, lactate dehydrogenase; CPK, creatine phosphokinase; T3, triiodothyroxine; T4, thyroxin.
Fig. 3. Plasma cortisol concentrations (mg/dL) in nine ponies fed twice a day (09:00 and 18:00 h) at 140% (closed diamonds) and 75% (open triangles) of their requirements. The oral glucose tests were performed on days 7 and 20 (indicated by the arrows). Data are summarised as means ± SE and significant difference (P < 0.05) are indicated by different letters.
Glucose (mg/dL)
Fig. 1. Bodyweight (means ± SE) in kg in nine ponies fed twice a day (09:00 and 18:00 h) at 140% (closed diamonds) and 75% (open triangles) of their requirements.
160 * *
120 *
*
80 40 0 0
60
120
180
240
300
Time after glucose load (min) Fig. 4. Mean plasma glucose concentration (mg/dL) of nine ponies after an oral glucose tolerance test performed after 7 days overfeeding (closed diamonds) and underfeeding (open triangles). Data are summarised as means ± SE and significant difference between feeding regimes at a similar time point are indicated with * for P < 0.05.
Ir Leptin (ng/mL)
4 ab
3
b
a b
b
2
b bc
c d d
d
d
cd
d
1 0 0
4
8
12
16
20
24
28
Time (Days) Fig. 2. Plasma leptin concentrations (ng/mL) in nine ponies fed twice a day (09:00 and 18:00 h) at 140% (closed diamonds) and 75% (open triangles) of their requirements. Data are summarised as means ± SE and significant difference (P < 0.05) are indicated by different letters.
ing feed restriction, plasma leptin concentration significantly decreased (P < 0.001). In general, plasma cortisol concentrations were higher during the underfeeding than during the overfeeding period, 26.0 ± 9.7 and 17.1 ± 4.7 mg/dL, respectively (P = 0.006) (Fig. 3). However, during the overfeeding period,
plasma cortisol concentrations were also elevated on days 7, 9 and 11, compared with days 1, 3 and 5 (P < 0.05). On day 20, when the oral glucose test during underfeeding took place, plasma cortisol levels of all ponies were extremely elevated (P < 0.001) and significantly higher (P = 0.009) compared to day 7 (the oral glucose test during overfeeding). During the OGTT, underfeeding was associated with a higher glucose concentration 180, 240 and 300 min after glucose administration (P < 0.05) (Fig. 4). AUC for glucose (P = 0.02) was significantly affected by treatment (P = 0.02) (29 ± 5 and 34 ± 7 g min dL-1 for over- and underfeeding, respectively). There was a trend towards a higher AUC for insulin during underfeeding (P = 0.05) (27 ± 16 and 45 ± 29 U min L 1 for over- and underfeeding, respectively). Discussion Bodyweight was not affected in any of the ponies so the results reported in this study were solely caused by changes
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in energy balance itself. Increased plasma NEFA concentrations during feed restriction are likely to be the result of an increased fatty acid mobilisation. However, parameters of metabolic rate, plasma T3 and T4 were not significantly changed, probably due to the short period of underfeeding (3 weeks) and the modest change in energy intake (75% of maintenance). Overfeeding in ponies resulted in a significant increase in leptin without changes in fat mass, as seen in humans (Kolaczynski et al., 1996). Most remarkable was that leptin concentrations returned to initial values within 7 days despite constant overfeeding. Changes in leptin may be explained by the lipostatic theory of bodyweight maintenance (Kennedy, 1953), which suggests that animals are able to maintain their bodyweight by adapting food intake and energy expenditure to available food (Buff et al., 2006) and energy requirements (Gordon et al., 2007). Besides reporting the status of body energy stores to the brain, leptin also functions as a sensor for energy balance. Hence, leptin is believed to play a key role in the lipostatic theory. In response to a positive energy balance, leptin will both reduce feed intake and increase metabolic rate in order to prevent changes in bodyweight. Additionally, leptin prevents further expression of the leptin gene due to a negative feedback mechanism and returns to initial concentrations (Houseknecht et al., 1998). In our study, underfeeding resulted in a decreased leptin concentration. Buff et al. (2006) also described a decrease in leptin in obese ponies receiving 75% of their ad libitum intake, and to a larger extent than in the present study. Placing the catheters before the OGTT resulted in elevated plasma cortisol concentrations (P < 0.05). However, plasma cortisol concentrations were higher during the second OGTT (P = 0.009), when ponies were in a negative energy balance (underfeeding). The effect of energy restriction on cortisol concentrations in literature is not clear. In contrast to the decreased cortisol concentration reported after prolonged nutrient restriction (Glade et al., 1984; Sticker et al., 1995), increased cortisol concentrations were found with hypoglycemia and acute feed restriction in horses (James et al., 1970) and donkeys (Forhead and Dobson, 1997). A clear interaction between energy status and plasma cortisol was therefore seen in our study. Catheter placement led to a higher cortisol increase during underfeeding than during the period of overfeeding. Considering that no elevated plasma cortisol concentrations were seen after consecutive OGTTs in other experiments using these same ponies (Van Weyenberg et al., unpublished data), it can be postulated that feed restriction was the main cause of the cortisol increase. During the OGTT, underfeeding was associated with a higher AUC for glucose (P = 0.02) and a trend for a higher AUC for insulin (P = 0.05). Consequently the higher glucose response during a negative energy balance was not the result of a decreased insulin secretion, but of a decreased glucose tolerance (AUC glucose/AUC insulin; P = 0.008). Diets higher in starch and sugar and lower in fibre, as fed
during overfeeding in this study, have been associated with insulin resistance (Hoffman et al., 2003). Thus, decreased glucose tolerance, seen during underfeeding was not caused by nutrient differences between the two periods and this higher glucose response could be explained by higher cortisol concentrations (Ralston, 2002). Conclusions Differences in feed intake and energy balance, separate from bodyweight changes, can influence plasma leptin concentrations and glucose tolerance in ponies. In addition, plasma leptin concentrations also changed as a function of the duration of the negative energy balance. For this reason both energy balance and the time after changed nutritional intake must be taken into account when leptin is used as predictor for fat mass or IR. Acknowledgements We thank Steven Galle for his excellent care of the ponies, Dirk Adriaensen for his help during the blood sampling and Herman De Rycke and Inge Vaesen for the analyses. References Buff, P.R., Johnson, P.J., Wiedmeyer, C.E., Ganjam, V.K., Messer, N.T., Keisler, D.H., 2006. Modulation of leptin, insulin, and growth hormone in obese pony mares under chronic nutritional restriction and supplementation with ractopamine hydrochloride. Veterinary Therapeutics 7, 64–72. Buff, P.R., Morrison, C.D., Ganjam, V.K., Keisler, D.H., 2005. Effects of short-term feed deprivation and melatonin implants on circadian patterns of leptin in the horse. Journal of Animal Science 83, 1023– 1032. Cartmill, J.A., Thompson, D.L., Storer, W.A., Gentry, L.R., Huff, N.K., 2003. Endocrine responses in mares and geldings with high body condition scores grouped by high vs low resting leptin concentrations. Journal of Animal Science 81, 2311–2321. Centraal Veevoederbureau, 1996. Het definitieve VEP- en VREp-systeem. CVB-rapport 15, pp 39–50. Darras, V.M., Visser, T.J., Berghman, L.R., Kuhn, E.R., 1992. Ontogeny of Type-I and Type-Iii deiodinase activities in embryonic and posthatch chicks – relationship with changes in plasma triiodothyronine and growth-hormone levels. Comparative Biochemistry and Physiology A-Physiology 103, 131–136. Dubuc, G.R., Phinney, S.D., Stern, J.S., Havel, P.J., 1998. Changes of serum leptin and endocrine and metabolic parameters after 7 days of energy restriction in men and women. Metabolism-Clinical and Experimental 47, 429–434. Forhead, A.J., Dobson, H., 1997. Plasma glucose and cortisol responses to exogenous insulin in fasted donkeys. Research in Veterinary Science 62, 265–269. Gentry, L.R., Thompson, D.L., 2002. The relationship between body condition, leptin, and reproductive and hormonal characteristics of mares during the seasonal anovulatory period. Theriogenology 58, 3– 566. Gentry, L.R., Thompson, D.L., Gentry, G.T., Davis, K.A., Godke, R.A., 2002. High versus low body condition in mares: Interactions with responses to somatotropin, GnRH analog, and dexamethasone. Journal of Animal Science 80, 3277–3285.
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