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METABOLISM AND NUTRITION |
* Center of Excellence for Poultry Science, University of Arkansas, Fayetteville 72701; and Growth Biology Laboratory, USDA-Agricultural Research Service, Beltsville, MD 20705
1 Corresponding author: ccoon{at}uark.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: breeder • feeding regimen • lipogenic gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of the problems associated with poor uniformity, skip-a-day (SKIP) feed-restriction programs are commonly used in preference to everyday (ED) programs. Feed-restriction can alter metabolic characteristics of the hen, such as its capacity for lipogenesis (Richards et al., 2003). In fact, Rosebrough and Steele (1985) and Rosebrough (2000) reported that intermittent feeding of broiler chickens resulted in an increase in lipogenic activity of the liver. Expression of several key lipogenic genes, such as acetyl-coenzyme A carboxylase (ACC; Hillgartner et al., 1996; Richards et al., 2003), malic enzyme (MAE), sterol regulatory element-binding protein-1 (SREBP-1), adenosine triphosphate-citrate lyase, fatty acid synthase (FAS), and stearoyl-coenzyme A (
9) desaturase-1 (SCD-1; Richards et al., 2003) genes, is elevated in restricted-fed compared with ad libitum-fed birds. Reports have also indicated that, at times, a disconnect exists between gene expression and enzyme activity. The level of mRNA does not always accurately predict the level of physiologically active enzyme within a cell. For example, Nur et al. (1995) revealed that starvation did not change glycogen synthase mRNA levels but did decrease enzyme activity, whereas Goodridge (1987) reported that feeding caused a rapid increase in FAS gene transcription prior to any increase in enzyme activity. To this end, changes in mRNA stability along with gene expression may also affect the levels of an enzyme (Richards et al., 2003).
Programs such as SKIP essentially represent a constant cycle of fasting and refeeding. During periods of fasting, certain tissues are able to mobilize reserves to satisfy the energy needs of the bird. Liver and adipose tissues are key sites of energy storage. Fatty acids are made available from adipose tissue and glucose is released from liver glycogen stores. During the refeeding period, liver glycogen stores are replenished and excess dietary energy is converted to triglycerides for storage.
Although the differences between restricted- and ad libitum-fed breeders have been described, little information is available regarding differences between types of feed-restriction programs. Furthermore, the time course changes in expression of lipogenic genes as affected by feed restriction are of great interest. Ma et al. (1990) reported that feeding fasted chickens stimulated MAE gene transcription in as little as 1.5 h following refeeding.
The aim of the study reported here was to determine what role different feed-restriction programs play in regulating hepatic lipid metabolism. The reactions to fasting and refeeding may differ between ED- and SKIP-fed pullets because of differences in the length of the fasting period. This study aimed to define those differences at several time points after feeding.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A flock of 350 Cobb 500 pullets was raised in 10 floor pens from 1 d old. The Cobb Breeder Management Guide (Cobb-Vantress, 2005) was used as a reference for all management conditions, including light schedules for dark-out rearing houses. The compositions of the diets used throughout the experiment are shown in Table 1
. Diets did not differ between treatments. The starter diet was fed from 0 to 4 wk of age and the grower diet was fed from 4 wk until the time of the experiment.
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The flock was fed ad libitum for the first 2 wk. From 2 to 4 wk, all birds were fed restricted amounts of feed every day. At 4 wk of age, feed-restriction treatments were implemented. Half the birds (5 randomly selected pens) were fed restricted amounts of feed every day from 4 to 16 wk of age (ED). Feed allocation was based on breeder recommended guidelines to reach target BW. The other half of the birds were fed every other day (SKIP). Total feed allocation did not differ between the 2 treatments. For example, if the ED group received 50 g per bird, the SKIP group would receive 100 g per bird every other day. Birds were weighed weekly by pen to adjust feed allocation to ensure that target BW was met.
At 108 d of age, 52 pullets from the ED group and 76 pullets from the SKIP group were randomly selected and individually caged. Cages (47 cm high, 30.5 cm wide, 47 cm high) were each equipped with an individual feeder and a water trough. Birds were fed individually and provided with free access to water at all times. The photoperiod was 8L:16D at this time. After 4 d of acclimation to individual cages (at 112 d), the 52 pullets from the ED group were fed 74 g of standard breeder grower diet (Table 1
), and the 76 pullets from the SKIP group were fed 148 g of the same diet at 7 a.m. Four pullets from each group were killed by CO2 asphyxiation immediately prior to feeding, and 4 pullets from each group were killed at several time points thereafter. The sampling intervals were 15 min, 30 min, 45 min, 60 min, 90 min, 2 h, and 4 h after initiation of feeding. From that point on, pullets were killed every 4 h. The last ED pullets were killed at 24 h after feeding, and the last SKIP pullets were killed at 48 h after feeding. This represented one entire feeding cycle for each of the groups. All pullets were weighed immediately after sacrifice and removal of crop contents. Livers from killed pullets were excised, weighed, and immediately frozen in liquid N for analysis of gene expression, enzyme activities, lipid content, and glycogen content.
Lipogenic Enzyme Gene Expression
Livers collected at 0, 12, 24, 36, and 48 h were used for gene expression analysis. Single-step reverse-transcription (RT)-PCR, on a real-time basis, was used to quantify mRNA expression of selected genes. Real-time RT-PCR detects amplification during early phases of reaction, whereas traditional PCR is measured at an end point (Bustin, 2000). In addition, real-time RT-PCR is potentially more sensitive because of a greater dynamic range (6 to 8 log) and an automated procedure for data analysis. Most installed software provides an output value known as C(t), which is the first calculated point of deflection from the baseline. The PCR cycle number corresponding to this value is indicative of gene expression. Real-time RT-PCR operates in the exponential part of the reaction curve and is thus limited by original template numbers.
Total RNA was isolated using the TriReagent procedure (Life Technologies, Rockville, MD) and measured spectrophotometrically and qualitatively by agarose gel electrophoresis. Duplicate RT-PCR reactions (25 µL) consisted of 1 to 2 µg of total RNA, 1x QuantiTect SYBR Green RT-PCR Master Mix (Invitrogen Corp., Carlsbad, CA), variable amounts of RNase water, QuantiTect RT mix, 2.5 mM Mg, and 0.5 µM each gene-specific primer, including a set for ß-actin, the internal standard. Reverse transcription proceeded for 30 min at 50°C in the presence of both Omniscript and Sensiscript (Qiagen, Valencia, CA). Initially, a 15-min incubation at 95°C was used to inactivate the RT reaction and to activate the HotStarTaq DNA polymerase (Qiagen). The following PCR cycle was repeated 45 to 50 times: denaturation for 15 s at 94°C, followed by annealing for 30 s at 58°C and extension for 30 s at 72°C. Fluorescence data were collected in the latter stage by noting SYBR Green incorporation into the extended DNA. Reverse-transcription PCR produced dsDNA amplicons of 423, 200, 447, 201, and 300 bp for FAS, MAE, ACC, aspartate aminotransferase (AAT), and ß-actin, respectively, using the primers described previously (Richards et al., 2003). Polymerase chain amplified product melting temperatures were determined by derivative calculations to determine homogeneity of amplified products. Aliquots of the RT-PCR reactions were loaded onto 1% agarose gels and electrophoresed at 10 V/ cm length of gel. Ethidium bromide was used to visualize DNA and orange G was used as the tracking dye. All amplicons were compared with a ladder composed of DNA standards of known size.
Fluorescence data were used to derive the C(t) or the PCR cycles to threshold, which was noted as the first significant deviation in fluorescence from a baseline value. The derived data were in log format and the relative mRNA levels were determined by subtracting the C(t) value of the gene of interest from the C(t) value of ß-actin. This difference became a power of 2, which was the relative difference between expression of the gene of interest and expression of ß-actin.
In Vitro Metabolism—Enzyme Assays
Part of the remaining liver tissues were homogenized (1:10, wt/vol) in 100 mM N-2-hydroyxethyl piperazine-N'-ethanesulfonic acid (HEPES, pH 7.5) and 3.3 mM ß-mercaptoethanol and centrifuged at 12,000 x g for 30 min (Rosebrough and Steele, 1985). The supernatant fractions were kept at –80°C until analyzed for the activities of MAE (EC 1.1.1.40 [EC] ), isocitrate dehydrogenase (ICDH, EC 1.1.1.42 [EC] ), and AAT (EC 2.6.1.1 [EC] ).
Malic enzyme activity was determined by a modification of the method of Hsu and Lardy (1969). Reactions contained 50 mM HEPES (pH 7.5), 1 mM nicotinamide adenine dinucleotide phosphate (NADP), 10 mM MgCl2, and the substrate, 2.2 mM L-malate (disodium salt), in a total volume of 1 mL. Portions (50 µL) of the 12,000 x g supernatants (diluted 1:10) were preincubated in the presence of the first 3 ingredients. Reactions were initiated by adding the substrate and following the rate of reduction of NADP at 340 nm at 30°C.
Isocitrate:NADP+ oxidoreductase-[decarboxylating] activity was determined by a modification of the method of Cleland et al. (1969). Reactions contained 50 mM HEPES (pH 7.5), 1 mM NADP, 10 mM MgCl2, and the substrate, 4.4 mM DL-isocitrate, in a total volume of 1 mL. Portions (50 µL) of the 12,000 x g supernatants (diluted 1:10) were preincubated in the presence of the first 3 ingredients. Reactions were initiated by adding the substrate and following the rate of reduction of NADP at 340 nm at 30°C.
Aspartate aminotransferase activity was determined by a modification of the method of Martin and Herbein (1976). Reactions contained 50 mM HEPES, 200 mM L-aspartate, 0.2 mM reduced nicotinamide adenine dinucle-otide, 1,000 units/L of malate:nicotinamide adenine dinucleotide+ oxidoreductase (EC 1.1.1.37 [EC] ) and the substrate, 15 mM 2-oxoglutarate, in a total volume of 1.025 mL. Portions (25 µL) of the 12,000 x g supernatants (diluted 1:20) were preincubated in the presence of the first 4 ingredients. Reactions were initiated by adding the substrate and following the rate of oxidation of reduced nicotinamide adenine dinucleotide at 340 nm at 30°C. Enzyme activities are expressed as micromoles of product formed per minute under the assay conditions (Rosebrough and Steele, 1985).
Liver Glycogen and Lipid Contents
Livers from 0-, 12-, 24-, 36-, and 48-h sampling periods were analyzed for glycogen with amyloglucosidase digestion (Keppler and Decker, 1974), followed by glucose residue analysis (Hill and Kessler, 1961). The remaining portions of all excised livers were frozen at –20°C until further processing. Livers portions were subsequently lyophilized in a Genesis SQ 12 EL freeze-drier (The VirTis Company, Gardiner, NY) to determine the DM content. Lyophilized samples were ground before further analysis. Liver ether extract was then analyzed according to AOAC (1990). Dry liver weight (LW) was obtained by multiplying the DM percentage by the wet LW. Liver fat percentage was determined on a DM basis. Total liver fat was obtained by multiplying dry LW by the percentage of fat in the dry liver sample.
Statistical Analyses
Data analysis was performed using JMP IN 5.1 (SAS Inst. Inc., Cary, NC) statistical analysis software. Chicks were assigned to treatments in a completely random manner. Data were analyzed using ANOVA. The statistical model included terms for feeding regimen and time after feeding. Data are presented as mean ± SEM. When effects were significant, means were separated using Tukey’s Studentized range test. All statements of significance are based on testing at P 
0.05.
Animal Use
All procedures were carried out in accordance with Animal Use Protocol No. 03008 for the experiment, which was approved by the University of Arkansas Institutional Animal Care and Use Committee.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mean BW, LW, and relative liver weight (RLW) from all birds over the 48-h experimental period are presented in Table 2. The RLW was calculated as (LW/BW) x 100. The mean BW of SKIP birds was lower (P 0.05) than that of ED birds. Both groups were allocated equal quantities of feed throughout their lifetimes. Therefore, the lower BW of SKIP birds is indicative of reduced efficiency of growth rather than differences in feed intake. Overall mean LW and RLW were higher (P 0.05) in SKIP-fed than in ED-fed pullets.
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. Relative LW for both ED- and SKIP-fed pullets were elevated (P 0.05) above the immediate prefeeding level by 8 h after feeding. The 8-h sampling point was the first point after the start of the dark period. Maximum RLW for ED pullets (2.43 g/100 g of BW) was reached at 16 h after feeding. Minimum RLW (1.63 g/100 g of BW) was observed 15 min after feeding in ED birds. The RLW of SKIP birds continued to increase until a peak (3.54 g/ 100 g of BW) was reached at 16 h after feeding. After this time, RLW declined to prefeeding levels by 28 h postfeeding. Minimum RLW for SKIP birds (1.99 g/100 g of BW) was observed at 40 h postfeeding. Maximum RLW was 50% higher in SKIP birds than in ED birds. The RLW of SKIP pullets was higher (P 0.05) than that of ED pullets at all time points except 45 min and 1 h.
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). The liver fat percentage was higher in ED birds than SKIP birds immediately prior to feeding, but total liver fat did not differ. Total liver fat was elevated (P 0.05) above immediate prefeeding levels by 12 h after feeding in both groups. In ED-fed birds, a peak in total liver fat (2.75 g) was reached 12 h after feeding. This was also the only time point at which total liver fat was significantly elevated from prefeeding levels. In SKIP birds, the peak in total liver fat (3.56 g) was reached at 24 h. Total liver fat was elevated (P 0.05) above prefeeding levels from 12 to 32 h after feeding. By 36 h, prefeeding total fat levels were restored. The maximum total fat content of SKIP pullets was 30% higher than that of ED pullets.
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0.05) from 0 (immediately prefeeding) to 12 h after feeding in ED pullets (Figure 3). By 24 h, prefeeding levels were restored. In SKIP pullets, liver glycogen was higher at 12, 24, and 36 h than at either 0 or 48 h. Maximum total glycogen was 2.55 g in ED pullets and 5.13 g in SKIP pullets. This represented a 2-fold increase in SKIP liver glycogen above ED liver glycogen.
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Acetyl-coenzyme A carboxylase gene expression relative to ß-actin for ED- and SKIP-fed pullets is presented in Figure 4. Expression of ACC was elevated (P 0.05) by 12 h after feeding in both groups. Expression of ACC in SKIP birds only reached a peak at 24 h after feeding but returned to prefeeding levels by 36 h. The maximum ACC expression in SKIP birds was more than 4-fold greater than the maximum in ED birds.
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. Expression of MAE was elevated in ED birds at 12 h, but this difference was not significant. Expression of MAE in SKIP birds was elevated (P 0.05) above the 0-h level at 12 and 24 h. The change in MAE expression from 0 to 24 h represented nearly a 60-fold increase. The magnitude of this elevation in MAE expression was greater than that for ACC. Expression of MAE in SKIP pullets returned to prefeeding levels by 36 h.
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) followed a trend very similar to that described for ACC. Immediately prior to feeding, FAS expression was higher in ED birds than in SKIP birds. Expression of FAS was highest at 12 h in ED birds but was not significantly (P 0.05) different from that at either 0 or 24 h. Expression of FAS was elevated (P 0.05) above prefeeding levels at 12 and 24 h in SKIP birds. By 36 h, expression of FAS was no longer higher than at 0 h. Expression of FAS reached a peak at 24 h in SKIP birds.
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). Although a minimum level of expression occurred at 12 h in ED birds, none of the differences between time points was significant (P 0.05). Expression of AAT was lower (P 0.05) at 12 h after feeding in SKIP birds but returned to prefeeding levels by 24 h.
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). Expression of ICDH at 0 h was higher (P 0.05) in SKIP birds than in ED birds. Expression of ICDH did not differ at 0, 12, or 24 h in ED birds. In SKIP birds, ICDH expression was lower (P 0.05) at 12 and 24 h than at 0 h. Levels returned to normal after 36 h. In the case of ICDH as well as AAT, 48-h gene expression in SKIP birds was elevated above all other time points.
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The enzyme activities of MAE, AAT, and ICDH were also measured (Figure 9). Activities of MAE, AAT, and ICDH did not differ (P 0.05) at 0, 12, or 24 h in ED-fed birds. In SKIP-fed birds, MAE activity was higher (P 0.05) at 24 h than at 0 or 12 h. Activity of AAT was lower (P 0.05) at 12 and 24 h than at 0, 36, or 48 h in SKIP-fed birds. Activity of ICDH was lower (P 0.05) at 12 and 24 h than at 0, 36, or 48 h in SKIP-fed birds.
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0.05) MAE activity at 0, 12, and 24 h than did ED birds at the same time. Activity of AAT did not differ between SKIP and ED birds at 0, 12, or 24 h. Activity of ICDH was higher in ED birds at 12 h than in SKIP birds at the same time, but did not differ at 0 or 12 h. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increased liver size in feed-restricted compared with ad libitum-fed birds has been noted previously (Muiruri et al., 1975; Rosebrough and Steele, 1985). In our study, RLW reached maximums in SKIP birds that were 50% higher than the maximums in ED birds. Muiruri et al. (1975) demonstrated in 2 experiments that RLW was close to 50% higher in meal-fed chicks 1 h after feeding than RLW in ad libitum-fed chicks. The dramatic changes in RLW of SKIP-fed pullets in our study are indicative of the dramatic changes in nutritional status that occur at 48-h intervals.
Muiruri et al. (1975) speculated that much of the increased LW after a meal was glycogen and water rather than lipids, and Leveille (1966) found nearly a 2-fold increase in liver glycogen concentration after refeeding of meal-fed chicks. In fact, our data suggest that there was a more than 5-fold increase in liver glycogen in SKIP-fed birds by 24 h after feeding. The increase after 12 h was closer to 2-fold in ED-fed birds.
Our results and those of other groups (Akiba et al., 1983; Katanbaf et al., 1989c) indicate that at least some part of the increased liver size after a meal is due to lipid accumulation. Total lipid levels increased approximately 6-fold in SKIP-fed birds by 24 h after feeding. In ED birds, the increase was approximately 3- to 4-fold by 12 h. Together, these data show that although glycogen does make the major contribution to increased LW after meal feeding, the contribution of lipids is significant. For ED pullets, liver fat increased by 1.97 g, whereas glycogen increased by 1.38 g from 0 to 12 h after feeding. In SKIP-fed pullets, liver fat increased by 2.96 g, whereas glycogen increased by 4.40 g from 0 to 24 h after feeding.
The liver is the major site of fatty acid synthesis in the chicken (O’Hea and Leveille, 1969; Hermier, 1997). Nutritional alteration of lipogenesis in birds can be achieved by alteration of energy and protein ratios (Yeh and Leveille, 1969; Donaldson, 1985; Rosebrough, 2000) and by fasting and refeeding (Yeh and Leveille, 1969; Rosebrough, 2000). It has been shown (Rosebrough and Steele, 1985; Rosebrough et al., 1988; Rosebrough, 2000) that fasting and refeeding of broiler chickens results in an increase in lipogenic activity of the liver. Although both ED and SKIP programs can be considered as meal-feeding situations, it was our contention that the severity of the fast and the size of the subsequent meal could potentially affect the known nutrient-lipogenic gene interactions.
Expression of ACC, MAE, and FAS genes was increased dramatically after feeding in SKIP birds. Although there were numerical increases in expression of MAE and FAS, only expression of ACC was significantly increased 12 h after feeding in ED birds. Richards et al. (2003) demonstrated that ACC, MAE, and FAS gene expression was elevated in feed-restricted birds. Other studies showed that feeding previously starved chicks with a high-carbohydrate, low-fat diet stimulated an 8- to 10-fold increase in hepatic ACC mRNA (Hillgartner et al., 1996). Muiruri et al. (1975) demonstrated a 50-fold increase in hepatic fatty acid synthesis after feeding of meal-fed chicks. The data presented in this report show very clearly that feeding regimens can have significant effects on hepatic metabolism, and that those effects are mediated by changes in expression of key lipogenic genes.
Increased lipogenesis in previously fasted, refed chickens can be ascribed to various factors, including increased hepatic ACC mRNA expression (Hillgartner et al., 1996; Richards et al., 2003), increased availability of reduced nicotinamide adenine dinucleotide phosphate (NADPH) reducing equivalents from the MAE reaction, and increased circulating triiodothyronine (T3) levels (Rosebrough, 2000). After refeeding, more glucose is available to the liver, providing more acetyl-coenzyme A via glycolysis as a substrate for lipogenesis. The rate-limiting step in lipogenesis occurs at ACC, which is regulated in the short term by covalent modification (phosphorylation) and allosteric control by citrate (Hillgartner et al., 1995, 1996). In the longer term, regulation of ACC occurs by transcriptional mechanisms, which are mediated by insulin, glucagon, T3, and glucose (Hillgartner et al., 1996). Nutritional factors can alter eventual enzyme activity by effects on transcriptional, posttranscriptional, and translational events (Hesketh et al., 1998). Feed restriction also increases the expression of other genes involved in lipogenesis. Richards et al. (2003) showed that expression of SREBP-1, adenosine triphosphate-citrate lyase, and SCD-1 genes was increased in feed-restricted (meal-fed) birds compared with ad libitum-fed birds. Sterol regulatory element-binding protein-1 is a transcription factor that directly influences the expression of other lipogenic enzymes (Richards et al., 2003). Those researchers found that SREBP-1 gene expression was positively correlated with MAE, CL, ACC, FAS, and SCD-1, reflecting the role of SREBP-1 as a key factor in coordinating lipogenesis.
Richards et al. (2003) also found correlations among levels of key metabolic hormones and expression of lipogenic genes. Both insulin and T3 induce genes coding for lipogenic enzymes as well as SREBP-1, whereas glucagon specifically inhibits the expression of SREBP-1 and lipogenic enzyme genes. In our study, it is likely that insulin and T3 levels were elevated in SKIP birds after feeding to levels above those of ED birds. Glucagon, on the other hand, likely increases in both ED and SKIP birds at the time when feed is no longer available, resulting in the observed reductions in lipogenesis as time after feeding increases. The crop can store large quantities of feed and provide a steady supply of feed to the gut for long periods, depending on the size of the meal. When the crop empties, changes in glucagon, blood glucose, and corticosterone likely combine to convert from an anabolic to a catabolic state.
Our study evaluated only changes in expression at 12-h intervals, but Rosebrough (2000) noted that increases in transcription could be seen as early as 1.5 h after refeeding. In this study, changes in MAE gene expression after feeding were of greater magnitude than those of ACC or FAS. This emphasizes the importance of the MAE reaction in providing reducing equivalents in the form of NADPH for lipogenesis. The inactivity of the pentose phosphate pathway in the chicken means that the role of MAE in lipogenesis is essential. The major fluctuations in the expression of ACC, MAE, and FAS genes in SKIP-fed chickens are indicative of the fluctuations in their nutritional status. On the other hand, the small alterations of ACC, MAE, and FAS gene expression in ED-fed birds reflect the much more consistent supply of nutrients in such feeding regimens. Rosebrough (2000) noted that the increase in MAE after cycles of starving and refeeding is a result of increased transcription and also improved mRNA stability. Richards et al. (2003) also emphasized the importance not only of increased transcription, but also of increased mRNA stability in mediating changes in lipogenic enzyme levels.
During the fasting period, birds must mobilize fatty acids to supply their energy needs. To maintain relatively steady levels of blood glucose, liver glycogen can be broken down and released as glucose. The supply of glucose from liver glycogen is quickly depleted however, as evidenced by the rapid decline in liver glycogen after 24 h in SKIP birds. Amino acid catabolism must supply the glucose deficit via gluconeogenesis. Isocitrate dehydrogenase is an enzyme that catalyzes the oxidative decarboxylation of isocitrate to 
-ketoglutarate and CO2 with NADP+ as cofactor. -Ketoglutarate is an intermediate in the citric acid cycle but also a precursor for the biosynthesis of certain amino acids. Aspartate aminotransferase catalyzes the reaction in which oxaloacetate accepts an amino group from glutamate to aspartate. The reaction requires -ketoglutarate to accept an amino group in a previous step. This fact may result in competition between ACC and the aconitase-isocitrate dehydrogenase pathway for cytoplasmic citrate. This is a potential mechanism by which increased dietary protein reduces ACC activity and lipogenesis (Rosebrough et al., 2002). The pattern of AAT and ICDH expression, relative to that of ACC, supports this theory.
In the study reported here, periods of fasting were associated with increased expression of ICDH and AAT genes. This is indicative of the increased gluconeogenesis occurring at such times. Expression of ICDH and AAT was not altered at different sampling times in ED-fed birds, whereas in SKIP-fed birds, expression of both genes was decreased after feeding and increased during the fasting period. Once again, these data are indicative of the more consistent nutrient supply in ED-fed birds. These data help to explain the lower efficiency of SKIP-fed birds. Deposition of nutrients after feeding and remobilization of those nutrients in the postabsorptive state is not a completely efficient process. This explains the lower BW in SKIP birds fed quantities of feed identical to those of ED birds. Several authors (Lee et al., 2001; Richards et al., 2003) have indicated that the role of ICDH in providing reducing equivalents for fatty acid synthesis is very limited. Rather, they have suggested a role for ICDH in cellular defense against oxidative damage. The expression patterns of MAE and ICDH noted in our study would also seem to indicate a limited role for ICDH in providing NADPH for fatty acid synthesis.
Levels of mRNA do not always accurately predict the amount of functional protein produced. In this study, enzyme activities of MAE, ICDH, and AAT were measured to determine whether changes in gene expression were related to changes in enzyme activity. We found that gene expression data accurately predicted the direction of changes in activity of MAE, ICDH, and AAT. However, the magnitude of observed changes in gene expression for SKIP-fed birds was far greater than the changes in enzyme activity. There are several potential posttranscriptional mechanisms by which such differences could be mediated. For example, Semenkovich et al. (1993) found that glucose availability could control FAS mRNA levels without having any effect on transcription initiation. They indicated that mRNA stability was affected by glucose. The occasional disparity between gene expression and enzyme activity was noted in studies on glycogen synthase (Nur et al., 1995), which showed that starvation did not change glycogen synthase mRNA but did decrease enzyme activity.
In summary, we showed that feeding regimens can have dramatic effects on nutrition-gene interactions. Whether the changes caused by SKIP feeding have any kind of effect on the subsequent production of these birds is unclear. Much of the increased lipid production is offset by the oxidation of that lipid during the fasting period, so changes in body composition may not be dramatic. Most producers switch all birds to ED feeding around the time of sexual maturity and onset of production. A study such as this one, conducted soon after switching to ED feeding, would help define the true effects of SKIP feeding on broiler breeder production. Understanding the links among nutrition, nutritional management, and gene expression will help nutritionists to better meet the needs of birds under very specific circumstances.
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ACKNOWLEDGMENTS |
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Received for publication February 17, 2007. Accepted for publication March 10, 2007.
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