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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Animal Sciences, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge 70803
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Key Words: Dexamethasone • Feeding • Insulin • Leptin • Stallions
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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; Elimam and Marcus, 2002) and cats (Appleton et al., 2002). In horses, McManus and Fitzgerald (2000) reported a decrease in plasma leptin in response to feed restriction in mares, whereas Piccione et al. (2004) reported that neither fasting nor physical activity had any effect on daily leptin patterns. We previously reported that plasma leptin concentrations increased in mares and geldings of high body condition following either a single injection (Cartmill et al., 2003b) or four daily injections (Gentry et al., 2002; Cartmill et al., 2003a) of dexamethasone (DEX). A similar increase in plasma leptin was observed in stallions following 5 d of DEX treatment (Cartmill, 2004). In the stallions, but not in mares or geldings, leptin concentrations varied diurnally, with peak concentrations occurring in the evening. This diurnal pattern in leptin concentrations was evident in both treated and control stallions, but was greatly enhanced by DEX treatment. A similar diurnal pattern in leptin concentrations was reported by Gentry et al. (2002) for mares in low body condition that were allowed to graze for only 2 h each morning. A common factor in these latter two experiments was the meal-fed nature of nutrient intake of the horses; stallions received a portion of their diet as a concentrate, fed each morning, and the thin mares were limit-grazed for 2 h, also in the morning. In contrast, the high-body-condition mares and geldings (Gentry et al., 2002; Cartmill et al., 2003a,b), which were free to graze throughout the day, did not exhibit diurnal changes in plasma leptin. The present experiments tested the hypotheses that daily cortisol rhythm, feeding time, and/or insulin infusion is the cause of the postprandial increase in plasma leptin concentrations in horses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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), were maintained in outdoor lots with minimal native grasses. They were provided Alicia bermudagrass hay and water ad libitum and were fed sufficient pelleted concentrate (approximately 1% of BW) once daily at 0700 to maintain body condition. The pelleted feed (Country Acres Horse Complete, Country Acres Co., Brentwood, MO) consisted of processed grain byproducts, roughage and forage products, plant protein products, molasses products, and mineral and vitamin mixes to achieve 12% (minimum) CP, 2.5% (minimum) crude fat, and 25% (maximum) crude fiber. The stallions were fed the pelleted feed and hay in the same manner throughout Exp. 1, 2, and 3. Experiment 1
Stallions were assigned randomly to receive either a single i.m. injection of DEX (125 µg/kg BW; n = 5; Sigma Chemical Co., St. Louis, MO) in vegetable oil or oil only (controls; n = 5) at 0700 on d –1. Beginning at 0700 the next day (0 h), blood samples were collected every 2 h for 36 h via jugular venipuncture into heparinized tubes to characterize hormonal changes throughout the day. The 24-h delay between DEX injection and onset of blood sampling was incorporated into the design, so that blood sampling would occur during a period of stimulated leptin secretion (Cartmill et al., 2003b).
Experiment 2
Two days after the conclusion of Exp. 1, the stallions were once again allotted to treatment groups for Exp. 2. Experiment 2 was designed to test the hypothesis that a 12-h shift in feeding time of the stallions would shift the leptin pattern in a similar manner. Five stallions were switched to feeding at 1900 daily, and the remaining five stayed on the 0700 feeding schedule. Previous results (Cartmill et al., 2003b) indicated that the effects of a single injection of DEX on leptin, glucose, insulin, and cortisol concentrations waned or were reversed by 10 d after injection; however, as a precaution, stallions were allotted to treatments with the restriction that each treatment group had two or three stallions that had previously received DEX in Exp. 1. After a 14-d adjustment period to the feeding regimens, blood samples were collected via jugular venipuncture every 4 h for 48 h beginning at 0700 on d 15; subsequent blood samples were collected at 0700 and 1900 for an additional 5 d.
Experiment 3
Approximately 12 mo after the conclusion of Exp. 2, six of the stallions were used in two 3 x 3 Latin square design experiments to test the hypothesis that insulin is the major factor responsible for the postprandial increase in plasma leptin concentrations. Treatments were 1) normal daily meal at 0700; 2) no feed for 24 h; and 3) no feed and a bolus injection of insulin (0.4 mIU/kg BW i.v.; Sigma) followed 2 min later by infusion of insulin at 1.2 mIU•kg BW–1•min–1 for 180 min, which was thereafter gradually decreased to 0 mIU•kg BW–1•min–1 by 240 min. Size of insulin bolus and amount of infusion were based on our previous experience with the hyperinsulinemic–euglycemic clamp technique (Cartmill, 2004; adapted from Powell et al., 2002). Sufficient glucose was infused throughout the insulin infusion to maintain euglycemia (monitored every 10 min). A Flo-Gard 6300 dual-channel volumetric infusion pump (I.V. Techonologies, Inc., Upperville, VA) was used for insulin and glucose infusion, and blood glucose concentrations were monitored with an Accuchek portable monitor (Roche Diagnostics Corp., Indianapolis, IN). Preliminary analysis of 107 blood samples via the portable monitor and the spectrophotometric method described below yielded the following regression equation: glucose via the portable monitor, mmol/L = 1.02 x glucose concentration via the laboratory method –0.24 (correlation coefficient = 0.802).
Treatment days were at least 7 d apart. On the days immediately before treatment days, stallions were placed in stalls in a barn and deprived of feed and hay beginning at 1500. On treatment days, blood samples were collected via jugular catheter at –60, 0, 10, 20, 40, and 60 min relative to start of treatment and then hourly for 24 h.
Laboratory and Statistical Analyses
In all experiments, blood samples were immediately centrifuged at 1,500 x g at 5°C and plasma was harvested and stored at –15°C. Concentrations of leptin in Exp. 1 and 2 were measured by RIA with commercial reagents (Linco Research Inc., St. Charles, MO; McManus and Fitzgerald, 2000; Cartmill et al., 2003a), whereas leptin was measured in Exp. 3 with an RIA developed and validated by Cartmill et al. (2003b). Cortisol and insulin were measured with commercially available RIA reagents (Diagnostic Systems Laboratory, Webster, TX). Assay sensitivities and intra- and interassay CV were 0.8 ng/mL, 4%, and 8%, respectively, for leptin via the Linco kits; 0.2 ng/mL, 6% and 4% for leptin, respectively, in Exp 3; 0.11 µg/dL, 5%, and 8%, respectively, for cortisol; and 2.0 µIU/mL, 5%, and 8%, respectively, for insulin. Concentrations of glucose were determined with a spectrophotometric assay (Method No. 315; Sigma).
Data from the three experiments were analyzed separately via the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) in a completely randomized design (Exp. 1), a randomized block design (Exp. 2) ANOVA, and a replicated Latin square design (Exp. 3), all with repetitive sampling (Gill and Hafs, 1971). The dependent variables were analyzed with horse, treatment, block (Exp. 2 only), day (Exp. 3 only), square (Exp. 3 only), time, and appropriate interactions included in the model. For Exp. 2 data, stallions were blocked based on previous treatment group in Exp. 1. Treatment, block, and their interaction were tested with the horse within treatment-block term. For Exp. 3 data, square, stallion within square, day within square, and treatment effects were tested with the square x horse x day x treatment interaction. In all experiments, time and its interaction with treatment were tested with residual error.
Differences between treatments at individual time points were assessed via the LSD test (Steel et al., 1997). In Exp. 1, due to heterogeneity of variances in cortisol and leptin concentrations after DEX injection, the data were first transformed to logarithms (base 10) for analysis. Leptin data for control stallions only also were analyzed separately in an ANOVA to test for the effect of time on leptin concentrations in the absence of DEX treatment; the effects of horse and time were tested with the horse x time interaction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Analysis of the untransformed cortisol data provided basically the same differences as the log-transformed data; thus, the untransformed means are presented. Concentrations of cortisol (Figure 1A) in control stallions varied (P < 0.01) with time, being greatest in the morning and least in the evening. Treatment with DEX suppressed (P < 0.01) cortisol concentrations. Concentrations of insulin (Figure 1B) were greater (P < 0.01) in DEX-treated stallions and increased (P < 0.01) in response to feeding in both DEX-treated and control stallions. The response to feeding in treated stallions was greater (P < 0.01) than that in control stallions. Concentrations of leptin (Figure 1C) in control stallions varied (P < 0.01) over time in an apparent diurnal pattern, peaking in the evening. This pattern of leptin secretion was similar, although of greater magnitude, in stallions treated with DEX (Figure 1D), and mean concentrations were greater (P < 0.01) in those stallions relative to controls 6 h after feeding and thereafter.
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Cortisol concentrations varied with time (P < 0.001; Figure 2A), and only isolated differences (treatment x time interaction; P = 0.02) occurred between groups. Cortisol concentrations in both groups were generally greatest in the mornings and least in the evenings and showed no indication of being shifted by the shift in feeding time. Plasma concentrations of glucose, insulin, and leptin (Figure 2B, C, D, respectively) all increased (P < 0.01) following feeding. There were interactions (P < 0.001) between treatment and time for insulin and leptin concentrations, and peak concentrations of both hormones in stallions fed at 0700 were generally different (P < 0.01) from corresponding concentrations in stallions fed at 1900.
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Insulin concentrations (Figure 3A) increased (P < 0.01) in stallions when they were meal-fed or infused with insulin and glucose, but insulin concentrations remained consistently low when stallions were not fed. The increase (and subsequent decrease) in insulin concentrations was earlier (P < 0.01) when insulin was infused compared with when the stallions were fed. Increased insulin concentrations were paralleled by gradual increases (P < 0.01) in leptin concentrations (Figure 3B) 3 to 4 h later when stallions were fed or infused with insulin and glucose. When stallions were not fed, leptin concentrations remained low throughout the 24-h period.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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; Miell et al., 1996; Ramsay and White, 2000) or in vitro (Cammisotto and Bukowiecki, 2002) are glucocorticoids and insulin. In our previous studies (Gentry et al., 2002; Cartmill et al., 2003a), DEX treatment greatly stimulated both leptin and insulin concentrations; thus, the two factors could not be separated in those experiments. In contrast, study of naturally occurring hyperleptinemia in obese mares and geldings (Cartmill et al., 2003b) revealed that those horses had elevated resting insulin concentrations as well as an exaggerated insulin response to glucose infusion, whereas daily cortisol concentrations and cortisol response to exercise were not different from those in normal obese horses. The present experiments were designed to clarify the role of insulin vs. glucocorticoids in the control of leptin secretion.
In Exp. 1, a single injection of dexamethasone to stallions suppressed plasma cortisol concentrations throughout the 36-h blood sampling period begun 24 h later. In previous reports (Slone et al., 1983; MacHarg et al., 1985; Cartmill et al., 2003b), treatment with DEX at this dose resulted in suppressed endogenous cortisol concentrations for 4 to 8 d. Thus, the glucocorticoid biological activity experienced by these treated stallions can be assumed to have been relatively high and constant during the 36 h of sampling that began on d 1. In contrast, control stallions exhibited the expected diurnal fluctuations in cortisol concentrations, with the greatest concentrations occurring in the morning and the least at night.
A diurnal pattern in plasma leptin concentrations was clearly seen in control stallions. Plasma leptin concentrations peaked approximately 10 h after feeding (which was at 0 and 24 h in Figure 1C
), then decreased to reach a nadir just before feeding the next morning. Dexamethasone treatment stimulated leptin concentrations and accentuated the diurnal pattern. Again, assuming a relatively constant glucocorticoid biological activity in the treated stallions during the 36-h sampling period, it was concluded that the diurnal pattern in leptin concentrations observed in both groups of stallions was not a result of diurnal patterns in glucocorticoid activity.
The diurnal pattern in leptin concentrations in stallions in Exp. 1 was similar to the 12-h variations in leptin reported by Gentry et al. (2002) for mares with low body condition that were grazed only 2 h daily each morning. In contrast, mares with high body condition allowed to graze most of the day (Gentry et al., 2002) and mares and geldings similarly kept on pasture (Cartmill et al., 2003a) did not exhibit diurnal variations in leptin secretion, neither under normal conditions nor after DEX treatment. Piccione et al. (2004) reported a "robust" daily rhythm of leptin concentrations in athletic as well as sedentary horses, whereas in that study, feed deprivation did not alter the diurnal pattern but did decrease average leptin concentrations. This is in contrast to our results in Exp. 3, in which leptin concentrations in stallions deprived of feed were consistently low for at least 36 h.
In a previous experiment, the effects of four daily injections of DEX at the dose used in Exp. 1 on cortisol, insulin, and leptin concentrations in mares and geldings waned within 14 d after the end of treatment (Cartmill et al., 2003a); thus, few carry-over effects were expected from Exp. 1 to Exp. 2. By assigning approximately half (two or three) of the previously DEX-treated stallions to each of the two treatment groups in Exp. 2, it was possible to block on this factor in the analyses of data in Exp. 2. In no case was the block effect a significant source of variation.
In Exp. 2, concentrations of insulin and leptin increased in response to feeding of the concentrate once daily, which is consistent with the results of Exp. 1, as well as with reports for other species (Dallongeville et al., 1998; Appleton et al., 2002; Elimam and Marcus, 2002). Similar increases in insulin concentrations have been reported for horses receiving pelleted feed on a once daily basis (DePew et al., 1994; Nadal et al., 1997). The secretion patterns of both insulin and leptin were shifted approximately 12 h when the time of feeding was shifted 12 h. The peak in insulin concentrations occurred 4 h after feeding in both feeding groups, and the insulin peak was followed by a peak in leptin concentrations 8 h later (12 h after feeding). When blood sampling was decreased to once every 12 h, remnants of the postprandial increase in both insulin and leptin (shifted 12 h in the two groups) were still apparent, even though the peaks were not being sampled. For comparison, peak concentrations of leptin in cats occurred 9 h after the insulin peak following a meal (Appleton et al., 2002). Similarly, a postprandial increase in insulin concentrations in sheep was followed 7 h later by increased plasma leptin (Marie et al., 2001). Marie et al. (2001) also reported a shift in both insulin and leptin peaks when time of feeding was delayed 4 h.
Results from other species have implicated the postprandial increase in insulin as a major factor in the diurnal pattern of leptin secretion. Koutsari et al. (2003) reported a strong, positive, linear relationship between postprandial insulin concentrations and postprandial leptin concentrations in healthy women. Moreover, in humans, treatment with insulin (Sivitz et al., 1998) and hyperinsulinemia (Cusin et al., 1995; Saladin et al., 1995) was reported to increase plasma leptin concentrations. Further, in vitro studies with cultured adipocytes have indicated a direct effect of insulin on increased leptin secretion (Ramsay and White, 2000; Cammisotto and Bukowiecki, 2002). In Exp. 3, i.v. infusion of insulin in stallions resulted in a postinfusion increase in leptin concentrations that was similar in magnitude and chronology to that observed after feeding. The insulin infusion rate was chosen based on our previous experiences with the hyperinsulinemic-euglycemic clamp technique (adapted from Powell et al., 2002; Cartmill, 2004). Although insulin concentrations increased faster in the infusion regimen (within minutes due to the priming injection) than after eating, the concentrations attained were within physiologic limits. Similarly, the increase and peak in leptin concentrations occurred earlier when stallions were infused with insulin than when they were fed. The conclusion that insulin caused the increase in leptin, even though glucose was infused at the same time, is supported by the fact that 1) blood glucose concentrations in Exp. 3 were not elevated during infusion but were maintained at normal concentrations, and 2) previous infusions of glucose at 200 mg/kg BW daily each morning for 4 d did not alter leptin concentrations 12 h later in geldings (Cartmill et al., 2003a).
In summary, treatment with DEX completely suppressed diurnal cortisol secretion in Exp. 1, but it enhanced the meal-fed response in both insulin and leptin concentrations. In Exp. 2, the diurnal patterns in cortisol concentration were not shifted by altering the feeding schedule by 12 h (Exp. 2), whereas the insulin and leptin peaks were shifted by 12 h. And in Exp. 3, elevation of insulin concentrations by insulin infusion, while maintaining euglycemia, resulted in increased leptin similar to that observed after meal feeding. These results are consistent with the hypothesis that feeding time, and more specifically the insulin increase associated with a meal, not cortisol rhythm, is responsible for the postprandial increase in plasma leptin concentrations in horses. In future experiments, the time at which blood samples are collected in relationship to consumption of a meal should be considered when comparing concentrations of leptin among horses.
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Footnotes |
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2 Current address: Texas A&M Agricultural Experiment Station, 3507 Hwy 59 E, Beeville, TX 78102.
3 Correspondence—phone: 225-578-3445; fax: 225-578-3279; e-mail: dthompson{at}agctr.lsu.edu.
Received for publication March 1, 2005. Accepted for publication April 29, 2005.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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