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The Journal of Nutrition Vol. 128 No. 4 April 1998,
pp. 677-682
Swine Research Group, Purina Mills, St. Louis, MO 63144 and * Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The relationship between obese gene expression and energy intake was determined in pigs of various body weights. With ad libitum consumption, expression increased (P < 0.001) with body weight from 55 to 163 kg. Obese mRNA relative abundance was correlated with fat mass (r = 0.74, P < 0.0001) and percentage of fat (r = 0.72, P < 0.0001). Obese expression was also evaluated at 159 kg (initial weight) and ad libitum, maintenance or 23% of maintenance intake for 28 d. Obese mRNA was independent of treatment (P > 0.78) despite considerable weight differences. Obese mRNA abundance was then compared at 136 kg (initial weight) and ad libitum or maintenance intake for 3 or 28 d. Abundance was not influenced by either duration of treatment or intake, despite a small increase (P < 0.01) in serum nonesterified fatty acids (NEFA) and a reduction (P < 0.02) in insulin attributable to maintenance intake. Finally, mRNA abundance was determined at 60 and 136 kg and conditions of food deprivation or ad libitum intake for 3 d. Food deprivation reduced (P < 0.01) serum insulin and increased (4- to 5-fold) NEFA concentrations. Obese mRNA abundance was greater (P < 0.01) in the heavier pigs and was reduced (P < 0.01) by food deprivation. We conclude that obese mRNA abundance in pigs correlates with fat mass and percentage of body fat under conditions of ad libitum intake. Furthermore, obese mRNA abundance is reduced by food deprivation, whereas lesser degrees of intake restriction do not change obese mRNA abundance, even when accompanied by appreciable weight loss.
KEY WORDS: obese gene · pigs · food intake · insulin · nonesterified fatty acids
Leptin, the product of the recently identified obese gene is a pleiotrophic peptide hormone that has been linked to the regulation of energy balance and body composition. Leptin is a peripherally derived signal, i.e, the obese gene is expressed only in adipose tissue (Bidwell et al. 1997 Obese mRNA abundance is higher in pigs with greater adipose mass (Bidwell et al. 1997 Animals and sample collection.
The pigs used in these studies were male castrates produced from parent stock (Camborough females mated to Line-63 males) obtained from the Pig Improvement Company (Franklin, KY). The pigs were reared in environmentally controlled buildings in individual pens and fed commercial-type diets that were nutritionally complete for the weight groups used. Calculated analyses of the diets used are presented in Table 1. At the conclusion of each study, final body weight and food intake measurements were collected and blood samples obtained by jugular venipuncture. Thereafter, adipose samples were collected by surgical biopsy (Experiments 3 and 4) or after the pigs were killed by exsanguination after mechanical stunning (Experiments 1 and 2). All adipose samples were collected from the subcutaneous depot located over the cervical spine. Briefly, for the biopsy, the pigs were restrained and the hair clipped from the biopsy site. The area was cleaned and disinfected with surgical scrub and local anesthesia (lidocaine, 2% injectable) applied. A circular punch biopsy, 2 cm in diameter and extending through the adipose layers to the underlying muscle, was removed and the site closed with a single cruciate suture. The biopsy site was covered with iodine spray and an antibiotic regimen administered. All sample collections were performed from ~1000 to 1130 h, 4-5.5 h after consumption of the morning meal by pigs with restricted intake. The middle and outer adipose layers were not separated for analysis. Rather, the sample from which total RNA was extracted for use in the ribonuclease protection assay was a composite sample representing both layers. All samples were stored at
Serum chemistry.
With the exception of insulin, serum variables were quantified using an automated clinical chemistry analyzer (Hitachi, Model 704, Indianapolis, IN) and colorimetric assay kits that are available commercially; glucose, triglyceride and urea nitrogen kits were purchased from Sigma Chemical (St. Louis, MO) and nonesterified fatty acid (NEFA) kits were purchased from Wako Chemical (Richmond, VA). Insulin was measured by RIA with the use of a kit purchased from Linco (St. Peters, MO).
Total RNA isolation.
Total RNA was extracted from all subcutaneous adipose tissue according to the method reported by Chomczynski and Sacchi (1987) Ribonuclease protection assay.
Because the abundance of obese mRNA is low in porcine adipose tissue, a sensitive ribonuclease protection assay was used for relative quantitative determinations. Construction of the transcription plasmid has been described previously (Bidwell et al. 1997
Experiment 1. Obese expression in pigs weighing 23-162 kg body weight.
Forty-eight male castrate pigs were selected from multiple litters at weaning and assigned at random (within litter) to be sampled at four selected weight targets. Final mean weights were 23, 55, 107 and 162 kg (n = 12). The pigs were killed as they reached their respective weight target (±2.5%). The gastrointestinal tract was removed, digesta expressed and the lumen washed gently with tap water. The empty body, including blood and viscera, was then ground and samples collected for submission to Ralston Analytical Laboratories (St. Louis, MO) for determination of chemical composition [protein (percentage of nitrogen × 6.25), fat (ether extract) and moisture (AOAC 1995)].
Experiment 2. Effect of energy intake and weight reduction on obese expression.
Twenty-four male castrate pigs were grown on a standard, nonlimiting dietary regimen to 159 kg body weight (± 2.5%) and assigned at random (n = 8) to three treatment groups on the basis of the degree of intake restriction. Consumption was ad libitum, restricted to the quantity of feed estimated for maintenance of body weight or restricted to 23% of the maintenance estimate to invoke an appreciable weight loss over the 28-d feeding period. The maintenance estimate for these pigs was predicted by using the Auspig3 model of swine growth. The daily allowance for both restricted groups (1500 and 350 g for maintenance and submaintenance, respectively) was divided into two equal rations fed at ~0700 and 1500 h.
Experiment 3. Effect of short- and long-term food intake restriction on obese expression.
Forty-eight male castrate pigs were grown on a standard, nonlimiting dietary regimen to 136 kg body weight (±2.5%) and then assigned at random (n = 12) to four treatments. The treatments were arranged as a 2 × 2 factorial with food consumption (ad libitum or maintenance) and duration of treatment (3 or 28 d) as the main effect variables. For the maintenance groups, the daily allowance (1350 g) was divided into two equal rations fed at ~0700 and 1500 h.
Experiment 4. Effect of food deprivation on obese expression.
Twenty-four male castrate pigs weighing 60 and 136 kg (± 2.5%) were assigned at random to two levels of food consumption (ad libitum or food deprived) in a 2 × 2 factorial arrangement of treatments (n = 12). The study lasted for 3 d.
Statistical analyses.
All data were analyzed by ANOVA (SAS 1997) for a randomized complete block design in which blocks were formed on the basis of pen arrangements within the housing facilities or replicate groups formed over time. Mean separations in Experiments 1 and 2 were accomplished by the least significant differences procedure. Experiments 3 and 4 were analyzed as a 2 × 2 factorial. Treatment df were partitioned to evaluate the main effects and their interaction via orthogonal contrasts.
Experiment 1.
Body composition, serum variables and obese mRNA data are presented in Table 2. Body fat (percentage and absolute quantity basis) increased (P < 0.01) at each body weight to a high of 29.3% (47.3 kg) at 162 kg. Under the conditions of ad libitum food intake used in this study, obese expression increased with body weight and adiposity. With the exception of the lightest two weight groups, obese expression was higher (P < 0.01) at each successive increase in body weight. Obese mRNA abundance was correlated with fat mass (r = 0.74, P < 0.0001) and percentage of fat (r = 0.72, P < 0.0001). From the lightest (23 kg) to the heaviest (162 kg) pigs, expression was increased 376%, an average of 8.3% for each kg of lipid deposited, with no apparent relationship to serum glucose or insulin status.
Experiment 2.
Body weight, serum chemistry and obese mRNA data are presented in Table 3. The changes in body weight were clearly reflective of the level of food intake. Over the 28-d experimental period, pigs allowed ad libitum consumption gained 14.5 kg, whereas the submaintenance group lost 22.3 kg (14.4% of their body weight). As expected, weight loss in pigs fed at the predicted maintenance level of intake was minimal (2.1%) and was likely caused in part by a reduction in gastrointestinal fill. Of the serum variables evaluated, only urea nitrogen and triglycerides were responsive to treatment. Urea nitrogen was 50-60% lower (P < 0.01) in the pigs allowed only maintenance or submaintenance levels of food consumption versus those allowed ad libitum intake. Serum triglycerides were 47% lower (P < 0.05) in pigs restricted to submaintenance intake. Serum insulin and glucose were not responsive to treatment (P > 0.39). Although there was a small increase in obese mRNA abundance in both intake-restricted groups vs. the group allowed ad libitum consumption, the difference was not significant (P > 0.73).
Experiment 3.
Body weight, serum chemistry and obese mRNA data are presented in Table 4. Weight change was influenced by intake level and the duration of the feeding period interactively (intake level × time, P < 0.01). Pigs allowed ad libitum consumption gained more weight than those restricted to maintenance consumption; the majority of this difference was attributable to the 28 d of ad libitum consumption. Weight change was negligible (
Experiment 4.
Weight change, serum chemistry and obese mRNA data are summarized in Table 5. Weight loss was influenced (P < 0.01) by both food deprivation and body weight. Weight loss was 66% greater in response to food deprivation in the heavier (136 kg) pigs than in the lighter (60 kg) group (body weight × intake level, P < 0.01). The heavier pigs had higher (P < 0.01) serum urea nitrogen concentrations than did the lighter group. Food deprivation resulted in lower (P < 0.01) urea nitrogen; this decrease was numerically greater in the heavier group (body weight × intake level, P < 0.20). Serum glucose was influenced only by body weight and was higher (P < 0.01) in the lighter pigs. Triglyceride concentrations were also higher (P < 0.01) in the lighter pigs, but this difference was largely the result of the differential response to food deprivation in the lighter versus heavier pigs (body weight × intake level, P < 0.01). In the lighter pigs, food deprivation caused a 21% increase in the triglyceride concentration, whereas in the heavier group there was a 24% reduction. Serum NEFA concentrations were higher (P < 0.08) in the lighter pigs and markedly higher (~5-fold) in pigs deprived of food. The magnitude of the increase in NEFA attributable to food deprivation was ~20% greater in the lighter pigs (body weight × intake level, P < 0.11). Food deprivation resulted in lower (P < 0.02) serum insulin independent of body weight. Obese mRNA abundance in tissue from the heavier pigs was over twice that of the lighter pigs (P < 0.01), and food deprivation lowered obese mRNA abundance by ~31% (P < 0.01). There were no interactive effects of intake and body size on obese mRNA abundance.
The linear increase in obese mRNA with adipose mass and percentage of body fat (Experiment 1) is consistent with earlier work in humans in which obese expression was correlated positively with adipocyte hypertrophy and hyperplasia (Hamilton et al. 1995 We thank Peg Curran, Gerald Maupin, Lloyd Harfst, Kelvin Peters and JoAnn Rademacher for care of the animals and excellent implementation of the protocols. We also express our sincere appreciation to Joanne Kuske, Becky Losinski and Robyn Pelker for technical assistance in obtaining the tissue samples, performing the RNA extractions and conducting the nuclease protection assays. We are grateful to Lyn Perry for serum chemistry determinations and to Larry Reutzel for statistical analyses. We thank Karen Houseknecht for conducting the insulin determinations.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Zhang et al. 1994) that is secreted into the blood (Halaas et al. 1995) and actively transported across the blood-brain barrier by a high affinity, saturable system (Banks et al. 1996). Leptin binds receptors in the central nervous system (Lynn et al. 1996, Malik and Young 1996) and activates neurons in the ventrobasal hypothalamus and brainstem (Elmquist et al. 1997). Furthermore, the expression of the obese gene and neuropeptide Y, a potent stimulator of food intake, are inversely related (Sainsbury et al. 1996, Schwartz et al. 1996, Stephens et al. 1995). The integrated effect of leptin administration to obese mice is reduced food intake, increased energy expenditure and normalized body weight and adiposity (Campfield et al. 1995, Halaas et al. 1995, Pellymounter et al. 1995).
), and expression and plasma concentrations are higher in pigs selected for greater fat deposition (Yen et al. 1997). Obese expression and circulating leptin concentrations are also correlated positively with body fat in rodent models of obesity (Frederich et al. 1995, Maffei et al. 1995) and in obese humans (Maffei et al. 1995). However, the physiologic role(s) of leptin extends beyond satiety. Leptin has been shown recently to abrogate neuroendocrine responses to starvation (Ahima et al. 1996), to suppress acetyl-CoA carboxylase expression and activity (Bai et al. 1996) and to inhibit insulin-induced glucose transport and lipogenesis (Müller et al. 1997). Leptin has also been related to depletion of lipid stores (Chen et al. 1996). There is no information available at present regarding changes in obese mRNA abundance in pigs during periods of weight reduction or weight stasis caused by caloric intake limitation or in response to food deprivation. We established initially a range of body weights (and fat masses) over which obese gene expression increases linearly under normal growing conditions. We then conducted three additional experiments to evaluate changes in obese mRNA levels under conditions of normal weight gain (ad libitum food intake), weight stasis (maintenance intake) and weight loss (submaintenance intake), and in response to short-term food deprivation.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
80°C until required for RNA extraction. All procedures and experiment protocols were reviewed and approved by the Purina Mills Animal Care & Use Committee.
View this table:
Table 1.
Calculated analyses of diets fed to the pigs used in all experiments1
. Tissue was homogenized in 4 mol/L guanidinium thiocyanate followed by the addition of 0.1 volume of 2 mol/L sodium acetate (pH 5.0). The samples were extracted sequentially with water-saturated phenol and chloroform/isoamyl alcohol (24:1) and the aqueous fractions precipitated with isopropanol. After a second precipitation in ethanol, the RNA pellets were resuspended in 10 mmol/L Tris, 1 mmol/L EDTA (pH 8.0) and analyzed by spectrophotometry for quantification (A260) and qualitative (A260:A280) determinations.
). Radiolabeling of the riboprobe was accomplished by in vitro transcription with T7 RNA polymerase in the presence of 32P-UTP. The radiolabeled antisense RNA is protected (178-bp fragment) from RNAse digestion after overnight hybridization with porcine adipose RNA. The in vitro transcription and RNAse protection assays were accomplished with the use of a commercially available kit (Maxiscript T7 + RPA II, Ambion, Austin, TX). Ribosomal RNA (18S or 28S) was used as an internal marker to which obese mRNA was standardized for relative quantitative comparisons. Because the effect of treatment on rRNA abundance was not significant (P > 0.31) in any experiment, obese mRNA was evaluated on a per unit of rRNA basis. Because of the abundance of rRNA relative to obese mRNA, the specific activity of the rRNA probe was reduced by mixing labeled and nonlabeled probes. Typically, 10-20 µg total RNA was used for the protection assay and X-ray film was exposed overnight (12-16 h). A sample autoradiograph is shown in Figure 1. All autoradiographs were quantified with the use of an image analysis system and software purchased commercially [Interactive Technologies International (ITTI), St. Petersburg, FL].
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Fig 1.
Autoradiograph of ribonuclease protection assay for porcine obese mRNA (Experiment 2). Total RNA was extracted from subcutaneous adipose tissue and 20-µg hybridized with the porcine obese antisense ribonucleic acid probe. The size of the protected fragment is 178 bp. The 18S rRNA probe was obtained from Ambion, Austin, TX. The protected fragment is 78 bp. Lanes 1-6 are as follows: 1) undigested obese riboprobe; 2) undigested 18S riboprobe; 3) digested nonprotected obese and 18S riboprobes; 4-6) obese and 18S protected fragments obtained with total RNA from pigs allowed maintenance, submaintenance or ad libitum intake, respectively.
View this table:
Table 2.
Empty-body chemical composition of pigs at selected target weights and serum metabolite concentrations1
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 3.
Effect of food intake restriction on body weight change, serum metabolite concentrations, and obese mRNA abundance in pigs1
3.2 kg) in pigs restricted to maintenance intake, irrespective of time. Serum glucose was not altered by treatment. However, urea nitrogen and triglycerides were lower (P < 0.01) in the pigs limited to maintenance intake. Nonesterified fatty acid concentrations were higher (P < 0.01) in pigs restricted to maintenance intake (67 and 102%, respectively, for the 3- and 28-d groups). Serum insulin was lower (P < 0.01) in the maintenance group irrespective of time. Obese mRNA abundance was not altered by level of intake or the duration of treatment.
View this table:
Table 4.
Effect of short- and long-term food intake restriction on body weight change and
selected serum metabolite concentrations in pigs1
View this table:
Table 5.
Effect of short-term food deprivation on body weight change and serum metabolite concentrations in pigs of different size
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The greater obese mRNA abundance in the heavier pigs of Experiment 4 is consistent with results of a previous investigation with pigs (Bidwell et al. 1997). In both the previous and present studies, the increase in fat mass occurred concomitantly with increasing age and body weight of the pigs. Accordingly, we cannot say with certainty that age-related hormonal changes or other factors did not contribute to the greater obese mRNA abundance. It should be noted that obese mRNA abundance was also highly correlated with protein mass (r = 0.77, P < 0.0001) and final body weight (r = 0.79, P < 0.0001). This study suggests that the increase in obese mRNA associated with increased fat mass and other variables was not dependent upon higher blood concentrations of insulin or glucose.
) and the acceptance of carbohydrate intake as a major regulator of serum leptin concentrations (Jenkens et al. 1997), we anticipated that restricting food intake and the ensuing weight loss would cause a reduction in obese mRNA. Pigs allowed only submaintenance intake (Experiment 2) lost 22.3 kg, 14.4% of their initial body weight. Such weight loss would be expected to have a significant effect on fat mass. Despite the considerable differences in body weight between the pigs allowed ad libitum consumption (14.6 kg gain, 168.6 kg final body weight) and those losing an average of 22.3 kg (132.3 kg final body weight) as a result of intake restriction, obese expression was not greater in the ad libitum intake group. Similar results were obtained in the third experiment in that weight gain in the nonrestricted pigs was not associated with higher obese mRNA abundance relative to those that maintained their weight. Scholz et al. (1996) reported that long-term intake restriction uncouples serum leptin from fat mass such that a deficiency of leptin may develop relative to adiposity. The data presented herein indicate an uncoupling of obese expression from fat mass in pigs but maintenance of obese mRNA abundance despite marked weight loss.
, MacDougald et al. 1995, Mizuno et al. 1996a and 1996b). It is highly unlikely that the short duration of food deprivation (3 d) was sufficient to cause measurable reductions in fat mass and adipocyte size in either weight group. Thus, the reduction in obese mRNA was likely independent of a significant change in fat mass and indicates that obese gene expression is reduced by hormonal or other regulatory changes invoked by food deprivation. The absence of a differential response to food deprivation in the larger versus smaller pigs may indicate that the difference in degree of obesity was not as great as what might occur in rodents and humans.
). Thus, these authors and Flier and Elmquist (1997) proposed the primary physiologic role of leptin to be that of a signaling system to the brain that invokes an array of metabolic and neuroendocrine responses to starvation. That a reduction in obese mRNA in pigs was achieved with nothing less than complete food deprivation (i.e., starvation) seems to support the proposed role for reduced leptin concentrations in the physiologic adaptation to starvation.
). It has been shown recently that PPAR-
down regulates reporter gene expression when the reporter gene is driven by the mouse obese promoter (Hollenberg et al. 1997). Furthermore, the possibility of a regulatory role for NEFA themselves cannot be discounted (Rentsch and Chiesi 1996). Finally, increased concentrations of catecholamines brought about by the food deprivation may have had a direct effect on obese expression via induction of cAMP production; Slieker et al. (1996) have shown that dibutyryl cAMP decreases obese mRNA abundance, and concensus cAMP response element sequences have been identified in the 5
untranslated region domain of the human obese promoter (Gong et al. 1996).
) adds another tier to the regulation of leptin activity that requires consideration when evaluating the relationship of leptin to a given physiologic state.
ACKNOWLEDGMENTS
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FOOTNOTES |
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Manuscript received 22 May 1997. Initial reviews completed 1 July 1997. Revision accepted 8 December 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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and peroxisome proliferator-activated receptor- on the leptin promoter. J. Biol. Chem. 272: 5283-5290. This article has been cited by other articles:
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  S. Lkhagvadorj, L. Qu, W. Cai, O. P. Couture, C. R. Barb, G. J. Hausman, D. Nettleton, L. L. Anderson, J. C. M. Dekkers, and C. K. Tuggle Microarray gene expression profiles of fasting induced changes in liver and adipose tissues of pigs expressing the melanocortin-4 receptor D298N variant Physiol Genomics, June 10, 2009; 38(1): 98 - 111. [Abstract] [Full Text] [PDF]
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C. A. Lents, R. P. Wettemann, F. J. White, I. Rubio, N. H. Ciccioli, L. J. Spicer, D. H. Keisler, and M. E. Payton Influence of nutrient intake and body fat on concentrations of insulin-like growth factor-I, insulin, thyroxine, and leptin in plasma of gestating beef cows J Anim Sci, March 1, 2005; 83(3): 586 - 596. [Abstract] [Full Text] [PDF]
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J. M. Hsu, P. H. Wang, B. H. Liu, and S. T. Ding The effect of dietary docosahexaenoic acid on the expression of porcine lipid metabolism-related genes J Anim Sci, March 1, 2004; 82(3): 683 - 689. [Abstract] [Full Text] [PDF]
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J. L. Miner The adipocyte as an endocrine cell J Anim Sci, March 1, 2004; 82(3): 935 - 941. [Abstract] [Full Text] [PDF]
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S.-T. Ding, A. Lapillonne, W. C. Heird, and H. J. Mersmann Dietary fat has minimal effects on fatty acid metabolism transcript concentrations in pigs J Anim Sci, February 1, 2003; 81(2): 423 - 431. [Abstract] [Full Text] [PDF]
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