Leptin and Luteinizing
Hormone Concentrations in Pigs

 

C.S. Whisnant and R.J. Harrell

 

Summary

Ovariectomized gilts were either placed on full feed (FF) or restricted to one-third of the full feed amount (RST) for seven days. Blood samples were taken through jugular cannulas every 15 min. for 4 h at the beginning and the end of the seven day period. Then dietary treatments were reversed and seven days later samples were taken as before. Serum concentrations of leptin, insulin and luteinizing hormone (LH) were determined by radioimmunoassay. LH pulse frequency and mean serum leptin and insulin concentrations were lower (P<.01) in RST than FF gilts. Reversal of treatment reversed the patterns of hormone secretion. These results confirm previous observations that feed restriction can inhibit pulsatile LH secretion and suggest this may be related to changes in leptin and/or insulin secretion. In a second experiment, no circadian rhythm in leptin secretion was found in pigs.

 

Introduction

Leptin is a protein produced by adipose tissue that can act as a hormone (1). It has been shown to decrease feed intake in pigs (2, 3) and other species (1) and affect the secretion of several hormones including luteinizing hormone (LH) (4). Leptin levels increase with increasing body fat (5). Short-term changes in dietary intake such as fasting (5, 6) or increased caloric intake (7) can affect leptin secretion. Mao et al. (8) found that feed restriction of lactating sows decreased serum leptin concentrations. In contrast, Spurlock et al. (9) reported that fasting but not lesser degrees of feed restriction decreased leptin mRNA abundance in pigs.

 

Leptin is involved with the reproductive system as well. Leptin administration increased LH secretion and decreased age at puberty in mice and rats (10). In rats and humans leptin secretion displays a circadian rhythm (11, 12). The objectives of the current study were to determine the effect of feed restriction on serum leptin and LH concentrations in ovariectomized gilts and in a separate experiment to determine if leptin secretion exhibited a circadian variation in pigs.

 

Material and Methods

Experiment 1. Twelve gilts that been ovariectomized as part of another experiment were weighed and randomly allotted to either feed restriction (RST) or full fed groups (FF) (n = 6/ group). The FF group received three kg per day of a mixed corn-soybean ration designed to meet the nutritional requirements for finishing pigs (13). The RST group received one kg of the same ration. Both groups were fed twice daily with one-half of the ration provided at each feeding. Water was available ad libitum. Gilts were housed in individual crates throughout the experiment. After one week on treatment the gilts were weighed and treatments reversed. The gilts that were restricted and then returned to full feed were classified as a separate group (Refed, RF).

 

Jugular cannulas were placed in gilts at three times: at initiation of the experiment before treatment diets were started, after 6 days on treatment and again six days after the reversal of feed treatments. Blood samples were taken at 15 min intervals for four hours on the day after cannulation. Samples were allowed to clot and then were stored overnight at 4C, centrifuged and serum stored at –20 C until assayed.

 

Experiment 2. Six barrows (30 kg body weight) were moved into an environmentally controlled chamber and given one week to adapt. The room was on a 12L:12D lighting schedule. Cannulas were placed into the jugular veins and samples were collected hourly over a 24 hour period. Lights were used to collect samples during the dark period. Samples were placed at 4C immediately and stored overnight until centrifugation. Serum was stored at –20 C until assayed for leptin. Feed intake was monitored during the study as well.

 

All samples from experiment 1 were assayed in duplicate for LH using a previously validated porcine LH assay. Intra and inter-assay coefficients of variation (CV) for the LH assay were 6.1 % and 9.7%, respectively. Hourly samples from both experiments were assayed for leptin in duplicate using the Linco Multi-Species Leptin Assay XL-85 kit (Linco, St. Louis, MO) as described per directions of the manufacturer. Insulin concentrations were measured in hourly samples from experiment 1 using a commercially available kit specific for porcine insulin (PI-12, Linco). Non-esterified fatty acids (NEFA) were measured in a single sample from each gilt in experiment 1 using a commercially available kit (WAKO Chemicals, Richmond, VA).

 

Results and Discussion

Serum leptin concentrations were not different between groups at the initiation of the experiment. After 1 week the serum leptin concentrations of RF gilts were decreased compared to those of FF gilts (P < .02) (Table 1). Reversing the dietary treatment resulted in increased serum leptin in the gilts now receiving FF and decreased serum leptin in gilts now receiving RF (Table 1).  Serum insulin concentrations were reduced (P<.01) and serum NEFA concentrations were increased (P<.01) by feed restriction (Table 1). Refeeding increased serum insulin and decreased NEFA concentrations. Pulsatile LH secretion was affected by dietary treatment with FF gilts having a higher LH pulse frequency (P< .05) (Table 1) and higher mean serum LH concentrations (P<.05) than RST gilts.

 

Serum concentrations of leptin did not differ over time in the hourly samples collected. There was no evidence of any circadian variation with an average serum concentration of 3.7 ± 0.9 ng/ mL.

 

The present study demonstrated that restricting feed intake to gilts resulted in decreased serum leptin concentrations. These results are similar to those reported in other species including mice, rats and humans (1). Increased consumption of calories increased serum leptin concentrations in humans (7). Our data also agree with previous studies in pigs. Qian et al. (6) reported that fasting reduced serum leptin concentrations in ovariectomized gilts within 20 hours of feed deprivation. Mao et al. (8) reported that 50% feed reduction for seven days to lactating sows resulted in lower serum leptin concentrations. Refeeding restored serum leptin concentrations to previous levels in our study. Refeeding was not tested in the previous studies.

 

Table 1.  Means (± ) SE for body weight, LH pulse frequency, serum leptin, insulin and serum NEFA1  for gilts from full fed (FF), restricted (RST), and refed (RF) groups.

 

Parameter

 

Treatment 2

             FF                          RST                            RF

Weight change (kg) 3

 8.5 ± 2.6a

    -12.4 ± 2.7b

11.1 ± 3.1a

Leptin ng/mL

4.7 ± 0.9

 2.6 ± 0.6

 4.1 ± 0.5

Insulin micro IU/mL

12.5 ± 2.6a

  7.2 ± 2.9b

14.2 ± 3.1a

LH Pulses / 4 hours

 3.7 ± 0.5

 2.6 ± 0.3

 3.5 ± 0.4

NEFA microEq/L1

    104.1 ± 7.2a

187.3 ± 10.6b

115.0 ± 8.3a

1 NEFA, nonesterified fatty acids

2 Within a row, means lacking a common superscript differ (P<.05)

3 Amount of weight change from previous week

 

No circadian variation was found in leptin secretion in experiment two. These barrows exhibited very little change during the 24 hour period. Lights were turned on to collect the hourly samples and this may have disrupted a circadian rhythm. In humans, the nocturnal rise in leptin appears to be entrained to meal timing and related to increased insulin secretion because of food ingestion during the waking period (1). Feed was available to these barrows ad libitum.

 

LH pulse frequency was significantly lower in RST ovariectomized gilts. The present results suggest that this model is useful for the study of nutritional effects on LH secretion and possible relationship with metabolic hormones such as leptin and insulin.

 

Serum insulin concentrations were decreased and serum NEFA concentrations were increased by feed restriction as expected in the current study. These data are in agreement with previous results (9). Refeeding restored concentrations of both to previous levels. Although this study does not provide evidence for a direct effect of leptin on LH secretion, the results do suggest that the decreased LH secretion found in RF gilts is related to changes in leptin secretion. It has been postulated that increased leptin is an indicator of a positive energy balance and could signal the hypothalamic-pituitary system that there is sufficient energy for reproduction (14). Insulin and NEFA concentrations could also play a role in regulating LH secretion during periods of restricted feeding. Further research is needed into the relationship of leptin secretion and LH secretion in the pig.

 

Literature Cited

1.                  Sinha MK, Caro JF. Clinical aspects of leptin. Vitamins and Hormones 54:1-, 1998.

2.         Barb CR, Yan X, Azain MJ, Kraeling RR, Rampacek GB,  Ramsay TG.  Recombinant leptin reduces feed intake and stimulates growth hormone secretion in swine. Dom Anim Endocrinol 15:77-86, 1998.

3.         Barb CR, Barrett JB, Kraeling RR, Rampacek GB. Role of leptin in modulating neuroendocrine function: A metabolic link between the brain-pituitary and adipose tissue. Reprod Dom Anim 34:111-125, 1999.

4.         Nagatani S, Guthikonda P, Thompson RC, Tsukamura H, Maeda K-I, Foster DL. Evidence for GnRH regulation by leptin: Leptin administration prevents reduced pulsatile LH secretion during fasting. Neuroendocrinology 67:370-376, 1998.

5.         Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 395:763-770, 1998.

6.         Qian H, Barb CR, Compton MM, Hausman GJ, Azain MJ, Kraeling RR, Baile CA. Leptin mRNA expression and serum leptin concentrations as influenced by age, weight, and estradiol in pigs. Dom Anim Endocrinol 16:135-143, 1999.

7.         Kolaczynski JW, Nyce MR, Considine RV, Boden G, Nolan JJ, Henry R, Olefsky J, Caro JF. Response of leptin to short-term fasting and refeeding in humans: a link with ketogenesis but not ketones themselves. Diabetes 45:1511-1515,1996.

8.         Mao J, Zak LJ, Cosgrove JR, Shostak S, Foxcroft GR. Reproductive, metabolic and endocrine responses to feed restriction and GnRH treatment in lactating primiparous sows. J Anim Sci 77:724-735, 1999.

9.         Spurlock ME, Frank GR, Cornelius SG, Ji Shaoquan, Willis GM, Bidwell CA. Obese gene expression in porcine adipose tissue is reduced by food deprivation but not by maintenance or submaintenance intake. J. Nutr. 128:677-682, 1998.

10.       Cheung CC, Thornton JE, Kuipjer JL, Weigle DS, Clifton DK, Steiner RA. Leptin is a metabolic gate for onset of puberty in the female rat. Endocrinology 138:855-858, 1997.

11.       Sinha MK, Ohannesien JP, Heiman ML, Kricauciunnas A, Stephens TW, Marco C, Caro JF. Nocturnal rise of leptin in lean, obese, and non-insulin dependent diabetes mellitus subjects. J. Clin. Invest. 97:1344-1347, 1996.

12.       Pickavance L, Tadavyon M, Willaims G, Vernon RG. Lactation suppresses diurnal rhythm of serum leptin. Biochem. Biophys. Res. Commun. 248:196-199, 1998.

13.       NRC. Nutrient Requirements of Swine (10th Ed.). National Academy Press, Washington, DC, 1998.

14.             Barash IA, Cheunh CC, Weigle DS, Ren H, Kabiting EB, Kuijper JL, Clifton DK, Steiner RA. Leptin is a metabolic signal to the reproductive system. Endocrinology 137:3144-3147, 1996.