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The Effect of Feed Restriction on Plasma Ghrelin, Growth Hormone, Insulin, and Glucose Tolerance in Pigs
The Professional Animal Scientist 26 (2010):26–34
©2010 American Registry of Professional Animal Scientists
Theon Effect of Feed Restriction Plasma Ghrelin, Growth
Hormone, Insulin, and Glucose Tolerance in Pigs R. Barretero-Hernandez,* M. L. Galyean,* PAS, and J. A. Vizcarra†1 *Department of Animal and Food Sciences, Texas Tech University, Lubbock 79409; and †Department of Food and Animal Sciences, Alabama A&M University, Normal 35762
ABSTRACT Twelve crossbred barrows (82.1 ± 1.1 kg BW) were assigned randomly to 2 diets to characterize ghrelin concentration and the metabolic profile of pigs undergoing dietary restriction. Pigs in one treatment group were allowed unrestricted access to feed (FF), whereas pigs in the other treatment group (RES) were fed 50% of the quantity of feed per unit of BW given to FF pigs. Animals in both treatments were fed daily (1 meal) beginning at 0900 h. Plasma samples were obtained twice weekly for 7 consecutive weeks to evaluate concentrations of ghrelin, growth hormone (GH), glucose, and insulin. Within each week, blood samples were taken before and 1.5 h after feeding. Three barrows in each dietary treatment were selected randomly for a glucose tolerance test before and after treatments were applied. The RES treatment decreased ADG and was associated with increased GH and decreased insulin concentrations. Glucose and insulin concentrations were decreased during fasting compared with concentrations 1.5 h after feeding. Results of the glucose tolerance test demonstrated increased glucose clear1 Corresponding author: jorge.vizcarra@ aamu.edu
ance rate, decreased glucose half-life, and decreased area under the curve in RES vs. FF pigs, reflecting the inability of FF animals to dispose of a test dose of glucose with the same efficiency as RES pigs. Restricting feed intake significantly altered glucose clearance rate, increased circulating concentrations of GH, and decreased insulin concentrations. Although restriction did not significantly affect plasma ghrelin, preprandial concentrations of ghrelin were increased compared with concentrations 1.5 h after feeding. Key words: pig, diet, feed restriction, ghrelin, glucose tolerance
INTRODUCTION Feed restriction in pigs can be used as a management tool to increase feed efficiency and nutrient utilization by taking advantage of compensatory growth during refeeding (Fabian et al., 2004; Kamalakar et al., 2009). In addition, feed restriction often occurs as a normal part of animal production systems, as well as during environmental stress or disease. For instance, out-of-feed events are associated with lower daily gain in swine production systems (Brumm et al., 2008), and voluntary feed intake is significantly
decreased in pigs exposed to high ambient temperatures (Collin et al., 2001). The metabolic and hormonal changes in the neuroendocrine axis during feed restriction in pigs are not completely understood; however, it has been demonstrated that growth hormone (GH) and insulin are involved in the homeostatic control of metabolism during feed restriction (Vandergrift et al., 1985; Buonomo and Baile, 1991; Hornick et al., 2000). More recently, the peripheral orexigenic signal ghrelin has been associated with the normal control of appetite in pigs (Salfen et al., 2004; Vizcarra et al., 2007; Dong et al., 2009). Ghrelin, a GH-releasing peptide, was first isolated from rat stomach while searching for an endogenous ligand to an “orphan” G-protein-coupled receptor (Kojima et al., 1999). Systemic exogenous administration of ghrelin induces adiposity in rodents by stimulating an acute increase in feed intake, as well as decreased fat utilization (Tschop et al., 2000; Wren et al., 2000; Nakazato et al., 2001). However, the orexigenic effect of peripheral ghrelin seems to be more potent in young, growing animals than in adult rats (Gilg and Lutz, 2006), and mice
that are null for the ghrelin receptor are not resistant to diet-induced obesity (Sun et al., 2008). In pigs, active immunization against ghrelin is associated with decreased voluntary intake (Vizcarra et al., 2007). In fact, ghrelin is the first and only known peripheral orexigenic signal (Bowers, 2001; Shintani et al., 2001; Muccioli et al., 2002; Horvath et al., 2003). A preprandial increase in plasma ghrelin has been observed in humans (Cummings et al., 2001), cattle (Hayashida et al., 2001), sheep (Sugino et al., 2004), rodents (Tschop et al., 2000), and pigs (Govoni et al., 2005). The predominant active form of ghrelin is n-octanoylated ghrelin. Octanoylation is a posttranslational process catalyzed by ghrelin O-acyl transferase, an enzyme that attaches n-octanoic acid to the amino residue Ser-3 (Gutierrez et al., 2008). The n-octanoyl moiety is essential for the activation of the GH secretagogue receptor type 1a for ghrelin. In fact, nonacylated ghrelin (des-acyl ghrelin) impairs receptor action or activation (Bednarek et al., 2000; Matsumoto et al., 2001). The octanoylated peptide is predominantly produced in the stomach within the oxyntic glands in rats and humans (Date et al., 2000; Sakata et al., 2002). Removal of the stomach in rats decreases serum ghrelin concentrations by 80%, suggesting that the stomach is the main source of ghrelin (Date et al., 2000; Dornonville de la Cour et al., 2001). Substantially lower amounts of ghrelin secretion and mRNA expression have been localized in several other tissues including other parts of the gut, the pituitary gland, and the hypothalamus (Gnanapavan et al., 2002; Cowley et al., 2003). The response of ghrelin, along with GH and insulin, to long-term feed restriction in pigs and the effects of feed restriction on the kinetics of glucose clearance have not been established. Thus, the objectives of this research were to evaluate changes in ghrelin, GH, glucose, and insulin concentrations during fasting and feed restriction, and to monitor glucose kinetics
Feed restriction and metabolites in pigs
27
after a glucose tolerance test (GTT) in feed-restricted growing pigs.
ments (wk 0), RES pigs were placed on the restricted feeding regimen for 5 consecutive weeks. The restricted percentage was applied based on the BW of each FF pig. For example, if pigs in the FF treatment consumed an average of 3% of their BW, each RES pig was offered 1.5% of its BW. As previously noted, fresh supplies of feed were offered daily at 0900 h. Body weight was recorded weekly (after 18 h of fasting) and was used to adjust the feeding level of pigs in the RES treatment group.
MATERIALS AND METHODS Treatments Twelve pigs with an average BW of 82.1 ± 1.1 kg were assigned randomly to 2 treatments (n = 6/treatment), 15 d before treatments were applied. Beginning at 20 wk of age (wk 0 of treatment), pigs in treatment group 1 were allowed unrestricted access to feed and water intake (FF). Pigs in treatment group 2 were placed in a restricted feeding regimen (RES) consisting of 50% of the quantity of feed per unit of BW given to pigs in the FF treatment and ad libitum water intake. In both treatment groups, pigs were fed daily (1 meal) beginning at 0900 h, using individual feeders. The diet given to FF and RES pigs was a corn- and soybean meal-based diet that met or exceeded current requirement estimates for pigs (NRC, 1998). On average, the diet contained 16.81% CP and 3,364 kcal/kg of ME. A detailed description of the diet used in the present experiment is described elsewhere (Ji et al., 2006). Blood samples were collected twice weekly, beginning on wk −2, for a total of 7 wk. On wk −1 and 5, a glucose tolerance test was conducted (described in a subsequent section). The animal care, treatment, and experimental protocols were approved by the Institutional Animal Care and Use Committee of Texas Tech University.
Weekly Blood Sampling Every Tuesday, blood was withdrawn twice during the day. Plasma samples were obtained before (fasting) and 1.5 h after feeding. The first sample was withdrawn after 18 h of fasting, and subsequently, all animals were fed according to their assigned treatments. The second sample was obtained 1.5 ± 0.5 h after feeding. Blood samples were collected via venipuncture in 5-mL evacuated tubes containing EDTA (Fisher Scientific, Pittsburgh, PA). Immediately after blood collection, aprotinin (500 kIU/mL of blood) was added to the collection tube to inhibit the activity of proteases. Tubes were gently rocked several times, placed on ice, and centrifuged (1,800 × g for 15 min) within 4 h. After centrifugation, plasma samples were labeled and stored at −80°C in cryogenic tubes until concentrations of ghrelin, GH, glucose, and insulin were analyzed as described in a subsequent section.
Feeding
Glucose Tolerance Test
Feed intake (as-fed basis) was measured daily for 7 wk, beginning 2 wk before treatments were applied. Daily feed intake was determined by recording the weight of feed offered each day minus any unconsumed feed remaining the next day. Average weekly feed intake was computed for statistical purposes. As noted previously, pigs were fed individually, and feeders were adjusted to minimize spillage. At the initiation of treat-
On wk −1 and 5, a GTT was conducted (n = 3 pigs/treatment). To facilitate blood collection and glucose infusion, pigs were cannulated the day before the GTT was administered, as described previously (BarreteroHernandez et al., 2009). After 18 h of fasting, blood samples (5 mL) were taken at 5-min intervals for 2 h beginning 10 min before glucose infusion. The glucose bolus (500 mg glucose/kg BW) was infused through the cannula
28 within 30 s. Sodium citrate (2.9% wt/vol) was used to flush cannulas between samples. Plain 12 × 75 mm tubes containing EDTA (57.0 μL; 0.5 M) were placed on ice before blood samples were obtained. After a blood sample was obtained, the tube was
Barretero-Hernandez et al.
gently rocked several times and centrifuged (1,800 × g for 20 min) within 4 h. Plasma samples were stored in labeled cryogenic tubes at −80 ± 2°C until glucose concentrations were determined.
Glucose and Hormone Assays Concentrations of ghrelin and GH were determined by RIA using commercial kits (for ghrelin: Phoenix Pharmaceuticals Inc., Belmont, CA; for GH: Linco Research Inc., St. Charles, MO), as described previously in pigs (Salfen et al., 2004; Vizcarra et al., 2007). Similarly, concentrations of insulin were determined by RIA using a solid-phase commercial kit (Coat-A-Count Diagnostic Products Corp., Los Angeles, CA), as described previously (Bossis et al., 2000). Plasma glucose concentrations were determined by an enzymatic colorimetric procedure (hexokinase-glucose dehydrogenase reagent kit, Pointe Scientific Inc., Canton, MI) using an automated microplate photometer (Multiskan Ascent, Thermo Fisher Scientific, Milford, MA).
Statistical Analyses
Figure 1. Average feed intake (A), BW gain (B), and G:F ratio (C) of pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs. Asterisks (*) indicate significant differences in feed intake, BW gain, and G:F for the given week (treatment × week interaction, P < 0.01).
Effects of treatment (FF vs. RES) on concentrations of ghrelin, GH, insulin, and glucose in weekly samples obtained after 18 h of fasting, and average feed intake, BW, and G:F were analyzed using repeated measurements over time (weeks). Similarly, the effect of sampling time (fasting vs. 1.5 h after feeding) on concentrations of ghrelin, GH, insulin, and glucose were analyzed using repeated measurements over time (MIXED procedure, SAS Inst. Inc., Cary, NC). At least 3 covariance structures were evaluated (compound symmetry, unstructured, and autoregressive). The autoregressive structure, with spatial power, provided the best model fit criteria. If a significant treatment × week or sampling time × week interaction existed, the SLICE option in SAS was used to test for a significant difference between treatments at each time. An exponential decay model of the form Y = βeγt was used to estimate glucose kinetics during the first 30 min of the GTT procedure (Freund and Littell, 2000) using the NLIN procedure in SAS. The parameter β represented the initial value of the re-
Feed restriction and metabolites in pigs
Figure 2. Growth hormone concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs. Asterisks (*) indicate significant differences in growth hormone for the given week (treatment × week interaction, P < 0.01).
sponse to glucose infusion at time (t) zero. In other words, β is an estimate of peak glucose concentration (G0). The glucose exponential decay rate was estimated by γ, and this parameter was used to derive the glucose clearance rate (clearance, %/min = 1 − eγ). The time required for glucose concentrations to fall by half (t1/2) also was derived from the exponential decay model [t1/2 = loge(2)/γ]. The exponential model was subsequently used to derive the approximate integral of total area under the curve (AUC) using the trapezoid method. Peak glucose concentrations, clearance rate, t1/2, and the AUC obtained from individual animals were analyzed as a completely randomized design. Glucose kinetics obtained from FF and RES animals on wk −1 (before treatments were applied) were not significantly different (P > 0.20), and data were pooled for statistical purposes.
kg/pig daily, whereas RES pigs consumed 1.6 ± 0.2 kg/pig daily. There was a treatment × week interaction for BW gain (P < 0.01). As expected, the RES treatment successfully induced a decrease in the
29 ADG (Figure 1B). On average, FF pigs gained 0.90 ± 0.07 kg/d after wk 2 compared with 0.25 ± 0.06 kg/d for RES pigs. In addition, there was a treatment × week interaction for the G:F ratio (P < 0.01; Figure 1C). The G:F ratio for RES pigs was decreased on wk 1, 3, and 4 when compared with FF pigs. Feed restriction at 50, 40, and 20% of ad libitum intake was also associated with decreased ADG in pigs (Vandergrift et al., 1985; McNeel et al., 2000; Lovatto et al., 2006). Feed restriction decreases nutrient intake and consequently modifies metabolic activity and the utilization of energy. The G:F ratio of FF animals was comparable with that previously reported in control barrows that were fed a similar diet (Ji et al., 2006), except for the G:F ratio obtained on wk 5 in the present experiment. The significant decrease in the G:F ratio
RESULTS AND DISCUSSION Feed Intake and Body Weight Dietary manipulation was used in pigs to mimic extreme BW changes and to evaluate associated changes in ghrelin, GH, insulin, and glucose kinetics. A treatment × week interaction was associated with a decrease in feed intake by RES pigs beginning on wk 1 (P < 0.001; Figure 1A). On average, FF pigs consumed 3.7 ± 0.2
Figure 3. A) Insulin concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs (treatment × week interaction; P < 0.01). B) Insulin concentration in pigs before (fasting) and after (+1.5 h) feeding. Values are shown averaged over the data for FF and RES pigs. Asterisks (*) indicate significant differences in insulin for the given week (sampling time × week interaction; P < 0.01).
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libitum for 5 wk), backfat thickness and adipocyte size was significantly decreased; however, no significant effects on the concentrations of anabolic transcripts associated with adipocytes were observed (McNeel et al., 2000). Taken together, it seems likely that the feed restriction in RES pigs signaled the GH neuroendocrine axis to increase GH concentrations.
Insulin and Glucose
Figure 4. A) Glucose concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs (P > 0.40). B) Glucose concentration in pigs before (fasting) and after (+1.5 h) feeding. Values are shown averaged over the data for FF and RES pigs. Asterisks (*) indicate significant differences in glucose for the given week (sampling time × week interaction; P < 0.01).
in RES animals can be attributed to the lower feed intake and decreased ingestion of amino acids (Veum et al., 1970; Chiba et al., 1991).
Growth Hormone There was a treatment × week interaction for GH concentrations (P < 0.01), which were increased for 4 consecutive weeks beginning on wk 2 of the experimental period in RES vs. FF pigs (Figure 2). No differences were observed in GH concentrations between fasting and 1.5 h after feeding (P > 0.20; data not shown). Growth hormone is secreted episodically in pigs (Clutter et al., 1995). In the present experiment, blood samples
were obtained at weekly intervals; therefore, an accurate estimation of pulsatile GH secretion was not feasible. Growth hormone is well known to play an important role in the regulation of metabolism, body composition, and energy expenditure. Undernutrition and dietary restriction induce a decrease in IGF-I and are associated with increased GH concentration in pigs (Kasser et al., 1981; Buonomo and Baile, 1991) and with GH resistance in most species (Hornick et al., 2000). It has been postulated that increased GH concentration during feed restriction allows fat mobilization to provide the additional energy needed to maintain homeostasis. When pigs were feed restricted (50% of ad
Insulin concentrations were increased in FF vs. RES pigs (treatment × week interaction, P < 0.01; Figure 3A); however, concentrations of glucose were not affected by treatment (Figure 4A). Given the significant differences in feed intake and the changes in insulin concentrations, reasons for the lack of response of glucose to treatment are not clear. Increased glucose concentration stimulates insulin release to facilitate uptake, utilization, and storage of glucose in muscle and adipose tissue (Kahn, 1996). Glucose transporters are activated when insulin binds to its plasma membrane receptors, allowing the cell to take up glucose and stimulate glycogen formation by activation of enzymatic systems. When glucose concentrations decrease, glucose transporters are recycled back into the cytoplasm, and glycogen synthesis stops (Shepherd and Kahn, 1999; Bryant et al., 2002). In the present experiment, insulin concentrations were increased in FF vs. RES pigs after 3 wk of treatment. Insulin promotes the uptake of fatty acids and the synthesis of lipids in the liver by increasing the availability of acetyl-CoA carboxylase and fatty acid synthase (Hillgartner et al., 1995). Changes in insulin, GH, and perhaps body fat mass in RES pigs may have resulted in changes in fatty acid metabolism. When lean and obese pigs were feed restricted (50% of ad libitum for 5 wk), backfat and fasting nonesterified fatty acid concentrations decreased in both lines (McNeel et al., 2000). In addition, fasting for 14 or 28 d significantly increased hepatic and renal gluconeogenesis and decreased
Feed restriction and metabolites in pigs
Figure 5. A) Ghrelin concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs (P > 0.20). B) Ghrelin concentration in pigs before (fasting) and after (+1.5 h) feeding. Values are shown averaged over the data for FF and RES pigs. There were significant sampling time and week effects on ghrelin concentrations (P < 0.01).
fatty acid synthesis and glucose oxidation in pigs (Klain et al., 1977). These data suggest that RES pigs had a lower lipolytic rate and enhanced generation of glucose from noncarbohydrate carbon substrates compared with FF pigs. Insulin and glucose concentrations were greater after feeding (+1.5 h) compared with concentrations after 18 h of fasting (sampling time × week interaction, P < 0.01; Figures 3B and 4B). This finding is consistent with the response observed in growing pigs, in which feed withdrawal for up to
48 h was associated with decreased insulin concentrations (Buonomo and Baile, 1991), reflecting the normal feedback mechanism devoted to maintain glucose homeostasis.
Ghrelin There were significant sampling time (P < 0.01) and week effects (P < 0.01) on ghrelin concentrations. On average, preprandial (fasting) ghrelin concentrations were 35.9 ± 0.9% greater than concentrations after feeding during the 7-wk experi-
31 mental period (Figure 5B). In gilts and weanling pigs, preprandial (72-h fasting) ghrelin concentrations were greater than those 6 to 48 h after feeding (Salfen et al., 2003; Govoni et al., 2005). Similar results have been reported in humans (Cummings et al., 2001), cattle (Hayashida et al., 2001), sheep (Sugino et al., 2004), and rodents (Tschop et al., 2000). In humans, plasma ghrelin concentrations rose almost 2-fold shortly before each meal and fell to base levels within 1 h after eating (Cummings et al., 2001). Additionally, administration of ghrelin to healthy human volunteers caused hunger sensations associated with increased plasma glucose followed by decreased insulin secretion (Broglio et al., 2001). Despite changes before and after feeding, concentrations of ghrelin were not significantly different (P > 0.20) between FF and RES pigs (Figure 5A). Systemic exogenous administration of ghrelin induces adiposity in rodents by stimulating an acute increase in feed intake, as well as a decrease in fat utilization (Tschop et al., 2000; Wren et al., 2000; Hayashida et al., 2001). However, as noted previously, the orexigenic effect of peripheral ghrelin seems to be more potent in young vs. adult rats (Gilg and Lutz, 2006), and mice that are null for the ghrelin receptor are subject to dietinduced obesity (Sun et al., 2008). In pigs, immunization against ghrelin is associated with decreased voluntary feed intake and decreased BW gain, providing evidence that ghrelin is involved in the long-term control of feed intake and BW in this species (Vizcarra et al., 2007). Peripheral infusion of ghrelin in 18-d-old pigs did not change feed intake; however, BW gain during the 5-d ghrelin infusion period was greater in treated piglets (Salfen et al., 2004). In the present study, ghrelin was responsive only to short-term feed withdrawal (18 h), but feed restriction for 5 wk was without effect. The lack of response of ghrelin to the 5-wk feed restriction program is perplexing because most of the published data suggest that ghrelin is involved in
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Table 1. Effect of feed restriction on glucose kinetics during a glucose tolerance test obtained on wk −1 and 5 in pigs that were allowed unrestricted access to feed and pigs that were fed 50% (BW basis) of the feed given to unrestricted pigs Week 5 Item
Week −11
FF2
G0 Clearance,5 %/min t1/2,6 min AUC7
373.4 1.78a 26.8a 5,166a
361.9 1.69a 26.9a 5,280a
4
a,b
a
RES3 a
SEM
359.9 2.47b 20.8b 3,150b a
14.5 0.17 1.5 604
Means within a row with different superscripts differ, P < 0.05.
1
Pooled data from all pigs before treatments were applied.
2
FF = full-fed pigs.
3
RES = pigs restricted to 50% (BW basis) of the feed provided to FF pigs.
4
G0 = peak glucose concentration.
5
Clearance rate per minute expressed as a percentage.
6
t1/2 = half-life.
7
AUC = area under the curve.
the long-term control of feed intake. Perhaps the length and degree of feed restriction imposed in the present experiment were not severe enough to produce a measurable effect on ghrelin secretion. Because only total ghrelin was measured, it is also possible that the variability observed in the present experiment is associated with differences between active and nonactive ghrelin forms. Further
research is needed to confirm and extend our results.
Glucose Tolerance Test All glucose kinetics obtained after the GTT procedures were significantly affected by treatment, except for peak glucose concentrations (Table 1). On wk 5, the glucose clearance rate was 62% greater in RES pigs than in FF animals. Consequently, the time
required for glucose concentrations to fall by half (t1/2) and the AUC were greater in FF than in RES pigs (Table 1; Figure 6). Insulin sensitivity was greater in RES animals compared with FF pigs on wk 5. Increased glucose clearance rate, decreased glucose t1/2, and decreased AUC during the GTT suggest that RES pigs were more responsive to endogenous insulin secretion in response to glucose infusion (Table 1; Figure 6). The glucose kinetics observed in FF pigs were similar to those reported in growing-finishing barrows consuming a diet similar to the one fed in the present experiment (Matthews et al., 2001). There is also good agreement of our data with those observed in humans. A decrease in BW resulting from restricted caloric intake or increased exercise improved glucose kinetics in obese patients (Gan et al., 2003; Weiss et al., 2006; Weiss and Holloszy, 2007). Taken together, our data suggest that the decreased tolerance for glucose in FF pigs on wk 5, as evidenced by longer t1/2 and greater AUC values compared with RES pigs, reflects the inability of the FF animals to dispose of a test dose of glucose with the same efficiency as RES pigs. In contrast, glucose kinetics in lactating sows were not affected by feed restriction (Quesnel et al., 2007). The differences between our results and those of Quesnel et al. (2007) probably reflect the degree of feed restriction, as well as differences in the physiological stage of the animals in the 2 studies. For example, glucose half-life is greater in pregnant sows than in nonpregnant animals (Pere et al., 2000). Nonetheless, the decrease of more than 6 min in glucose half-life in RES pigs at wk 5 can be deemed physiologically relevant because changes of the same extent were previously observed in pigs under contrasting physiological and nutritional treatments (Pere et al., 2000; Matthews et al., 2001).
IMPLICATIONS Figure 6. Average glucose concentrations during a glucose tolerance test (wk 5) in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs.
A significant preprandial increase in ghrelin was observed after fast-
Feed restriction and metabolites in pigs
ing; however, feed restriction for 5 consecutive weeks did not alter ghrelin concentrations, suggesting that plasma ghrelin concentrations are responsive only to short-term feed withdrawal. Alternatively, the length of time and severity of feed restriction imposed in the present experiment might not have been sufficient to produce measurable effects on ghrelin concentrations. Although ghrelin did not respond to feed restriction, feed-restricted pigs had increased GH and decreased insulin concentrations in plasma, and restricted pigs were more responsive to glucose clearance, half-life, and the AUC after glucose infusion than was the case with fullfed pigs.
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©2010 American Registry of Professional Animal Scientists
Theon Effect of Feed Restriction Plasma Ghrelin, Growth
Hormone, Insulin, and Glucose Tolerance in Pigs R. Barretero-Hernandez,* M. L. Galyean,* PAS, and J. A. Vizcarra†1 *Department of Animal and Food Sciences, Texas Tech University, Lubbock 79409; and †Department of Food and Animal Sciences, Alabama A&M University, Normal 35762
ABSTRACT Twelve crossbred barrows (82.1 ± 1.1 kg BW) were assigned randomly to 2 diets to characterize ghrelin concentration and the metabolic profile of pigs undergoing dietary restriction. Pigs in one treatment group were allowed unrestricted access to feed (FF), whereas pigs in the other treatment group (RES) were fed 50% of the quantity of feed per unit of BW given to FF pigs. Animals in both treatments were fed daily (1 meal) beginning at 0900 h. Plasma samples were obtained twice weekly for 7 consecutive weeks to evaluate concentrations of ghrelin, growth hormone (GH), glucose, and insulin. Within each week, blood samples were taken before and 1.5 h after feeding. Three barrows in each dietary treatment were selected randomly for a glucose tolerance test before and after treatments were applied. The RES treatment decreased ADG and was associated with increased GH and decreased insulin concentrations. Glucose and insulin concentrations were decreased during fasting compared with concentrations 1.5 h after feeding. Results of the glucose tolerance test demonstrated increased glucose clear1 Corresponding author: jorge.vizcarra@ aamu.edu
ance rate, decreased glucose half-life, and decreased area under the curve in RES vs. FF pigs, reflecting the inability of FF animals to dispose of a test dose of glucose with the same efficiency as RES pigs. Restricting feed intake significantly altered glucose clearance rate, increased circulating concentrations of GH, and decreased insulin concentrations. Although restriction did not significantly affect plasma ghrelin, preprandial concentrations of ghrelin were increased compared with concentrations 1.5 h after feeding. Key words: pig, diet, feed restriction, ghrelin, glucose tolerance
INTRODUCTION Feed restriction in pigs can be used as a management tool to increase feed efficiency and nutrient utilization by taking advantage of compensatory growth during refeeding (Fabian et al., 2004; Kamalakar et al., 2009). In addition, feed restriction often occurs as a normal part of animal production systems, as well as during environmental stress or disease. For instance, out-of-feed events are associated with lower daily gain in swine production systems (Brumm et al., 2008), and voluntary feed intake is significantly
decreased in pigs exposed to high ambient temperatures (Collin et al., 2001). The metabolic and hormonal changes in the neuroendocrine axis during feed restriction in pigs are not completely understood; however, it has been demonstrated that growth hormone (GH) and insulin are involved in the homeostatic control of metabolism during feed restriction (Vandergrift et al., 1985; Buonomo and Baile, 1991; Hornick et al., 2000). More recently, the peripheral orexigenic signal ghrelin has been associated with the normal control of appetite in pigs (Salfen et al., 2004; Vizcarra et al., 2007; Dong et al., 2009). Ghrelin, a GH-releasing peptide, was first isolated from rat stomach while searching for an endogenous ligand to an “orphan” G-protein-coupled receptor (Kojima et al., 1999). Systemic exogenous administration of ghrelin induces adiposity in rodents by stimulating an acute increase in feed intake, as well as decreased fat utilization (Tschop et al., 2000; Wren et al., 2000; Nakazato et al., 2001). However, the orexigenic effect of peripheral ghrelin seems to be more potent in young, growing animals than in adult rats (Gilg and Lutz, 2006), and mice
that are null for the ghrelin receptor are not resistant to diet-induced obesity (Sun et al., 2008). In pigs, active immunization against ghrelin is associated with decreased voluntary intake (Vizcarra et al., 2007). In fact, ghrelin is the first and only known peripheral orexigenic signal (Bowers, 2001; Shintani et al., 2001; Muccioli et al., 2002; Horvath et al., 2003). A preprandial increase in plasma ghrelin has been observed in humans (Cummings et al., 2001), cattle (Hayashida et al., 2001), sheep (Sugino et al., 2004), rodents (Tschop et al., 2000), and pigs (Govoni et al., 2005). The predominant active form of ghrelin is n-octanoylated ghrelin. Octanoylation is a posttranslational process catalyzed by ghrelin O-acyl transferase, an enzyme that attaches n-octanoic acid to the amino residue Ser-3 (Gutierrez et al., 2008). The n-octanoyl moiety is essential for the activation of the GH secretagogue receptor type 1a for ghrelin. In fact, nonacylated ghrelin (des-acyl ghrelin) impairs receptor action or activation (Bednarek et al., 2000; Matsumoto et al., 2001). The octanoylated peptide is predominantly produced in the stomach within the oxyntic glands in rats and humans (Date et al., 2000; Sakata et al., 2002). Removal of the stomach in rats decreases serum ghrelin concentrations by 80%, suggesting that the stomach is the main source of ghrelin (Date et al., 2000; Dornonville de la Cour et al., 2001). Substantially lower amounts of ghrelin secretion and mRNA expression have been localized in several other tissues including other parts of the gut, the pituitary gland, and the hypothalamus (Gnanapavan et al., 2002; Cowley et al., 2003). The response of ghrelin, along with GH and insulin, to long-term feed restriction in pigs and the effects of feed restriction on the kinetics of glucose clearance have not been established. Thus, the objectives of this research were to evaluate changes in ghrelin, GH, glucose, and insulin concentrations during fasting and feed restriction, and to monitor glucose kinetics
Feed restriction and metabolites in pigs
27
after a glucose tolerance test (GTT) in feed-restricted growing pigs.
ments (wk 0), RES pigs were placed on the restricted feeding regimen for 5 consecutive weeks. The restricted percentage was applied based on the BW of each FF pig. For example, if pigs in the FF treatment consumed an average of 3% of their BW, each RES pig was offered 1.5% of its BW. As previously noted, fresh supplies of feed were offered daily at 0900 h. Body weight was recorded weekly (after 18 h of fasting) and was used to adjust the feeding level of pigs in the RES treatment group.
MATERIALS AND METHODS Treatments Twelve pigs with an average BW of 82.1 ± 1.1 kg were assigned randomly to 2 treatments (n = 6/treatment), 15 d before treatments were applied. Beginning at 20 wk of age (wk 0 of treatment), pigs in treatment group 1 were allowed unrestricted access to feed and water intake (FF). Pigs in treatment group 2 were placed in a restricted feeding regimen (RES) consisting of 50% of the quantity of feed per unit of BW given to pigs in the FF treatment and ad libitum water intake. In both treatment groups, pigs were fed daily (1 meal) beginning at 0900 h, using individual feeders. The diet given to FF and RES pigs was a corn- and soybean meal-based diet that met or exceeded current requirement estimates for pigs (NRC, 1998). On average, the diet contained 16.81% CP and 3,364 kcal/kg of ME. A detailed description of the diet used in the present experiment is described elsewhere (Ji et al., 2006). Blood samples were collected twice weekly, beginning on wk −2, for a total of 7 wk. On wk −1 and 5, a glucose tolerance test was conducted (described in a subsequent section). The animal care, treatment, and experimental protocols were approved by the Institutional Animal Care and Use Committee of Texas Tech University.
Weekly Blood Sampling Every Tuesday, blood was withdrawn twice during the day. Plasma samples were obtained before (fasting) and 1.5 h after feeding. The first sample was withdrawn after 18 h of fasting, and subsequently, all animals were fed according to their assigned treatments. The second sample was obtained 1.5 ± 0.5 h after feeding. Blood samples were collected via venipuncture in 5-mL evacuated tubes containing EDTA (Fisher Scientific, Pittsburgh, PA). Immediately after blood collection, aprotinin (500 kIU/mL of blood) was added to the collection tube to inhibit the activity of proteases. Tubes were gently rocked several times, placed on ice, and centrifuged (1,800 × g for 15 min) within 4 h. After centrifugation, plasma samples were labeled and stored at −80°C in cryogenic tubes until concentrations of ghrelin, GH, glucose, and insulin were analyzed as described in a subsequent section.
Feeding
Glucose Tolerance Test
Feed intake (as-fed basis) was measured daily for 7 wk, beginning 2 wk before treatments were applied. Daily feed intake was determined by recording the weight of feed offered each day minus any unconsumed feed remaining the next day. Average weekly feed intake was computed for statistical purposes. As noted previously, pigs were fed individually, and feeders were adjusted to minimize spillage. At the initiation of treat-
On wk −1 and 5, a GTT was conducted (n = 3 pigs/treatment). To facilitate blood collection and glucose infusion, pigs were cannulated the day before the GTT was administered, as described previously (BarreteroHernandez et al., 2009). After 18 h of fasting, blood samples (5 mL) were taken at 5-min intervals for 2 h beginning 10 min before glucose infusion. The glucose bolus (500 mg glucose/kg BW) was infused through the cannula
28 within 30 s. Sodium citrate (2.9% wt/vol) was used to flush cannulas between samples. Plain 12 × 75 mm tubes containing EDTA (57.0 μL; 0.5 M) were placed on ice before blood samples were obtained. After a blood sample was obtained, the tube was
Barretero-Hernandez et al.
gently rocked several times and centrifuged (1,800 × g for 20 min) within 4 h. Plasma samples were stored in labeled cryogenic tubes at −80 ± 2°C until glucose concentrations were determined.
Glucose and Hormone Assays Concentrations of ghrelin and GH were determined by RIA using commercial kits (for ghrelin: Phoenix Pharmaceuticals Inc., Belmont, CA; for GH: Linco Research Inc., St. Charles, MO), as described previously in pigs (Salfen et al., 2004; Vizcarra et al., 2007). Similarly, concentrations of insulin were determined by RIA using a solid-phase commercial kit (Coat-A-Count Diagnostic Products Corp., Los Angeles, CA), as described previously (Bossis et al., 2000). Plasma glucose concentrations were determined by an enzymatic colorimetric procedure (hexokinase-glucose dehydrogenase reagent kit, Pointe Scientific Inc., Canton, MI) using an automated microplate photometer (Multiskan Ascent, Thermo Fisher Scientific, Milford, MA).
Statistical Analyses
Figure 1. Average feed intake (A), BW gain (B), and G:F ratio (C) of pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs. Asterisks (*) indicate significant differences in feed intake, BW gain, and G:F for the given week (treatment × week interaction, P < 0.01).
Effects of treatment (FF vs. RES) on concentrations of ghrelin, GH, insulin, and glucose in weekly samples obtained after 18 h of fasting, and average feed intake, BW, and G:F were analyzed using repeated measurements over time (weeks). Similarly, the effect of sampling time (fasting vs. 1.5 h after feeding) on concentrations of ghrelin, GH, insulin, and glucose were analyzed using repeated measurements over time (MIXED procedure, SAS Inst. Inc., Cary, NC). At least 3 covariance structures were evaluated (compound symmetry, unstructured, and autoregressive). The autoregressive structure, with spatial power, provided the best model fit criteria. If a significant treatment × week or sampling time × week interaction existed, the SLICE option in SAS was used to test for a significant difference between treatments at each time. An exponential decay model of the form Y = βeγt was used to estimate glucose kinetics during the first 30 min of the GTT procedure (Freund and Littell, 2000) using the NLIN procedure in SAS. The parameter β represented the initial value of the re-
Feed restriction and metabolites in pigs
Figure 2. Growth hormone concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs. Asterisks (*) indicate significant differences in growth hormone for the given week (treatment × week interaction, P < 0.01).
sponse to glucose infusion at time (t) zero. In other words, β is an estimate of peak glucose concentration (G0). The glucose exponential decay rate was estimated by γ, and this parameter was used to derive the glucose clearance rate (clearance, %/min = 1 − eγ). The time required for glucose concentrations to fall by half (t1/2) also was derived from the exponential decay model [t1/2 = loge(2)/γ]. The exponential model was subsequently used to derive the approximate integral of total area under the curve (AUC) using the trapezoid method. Peak glucose concentrations, clearance rate, t1/2, and the AUC obtained from individual animals were analyzed as a completely randomized design. Glucose kinetics obtained from FF and RES animals on wk −1 (before treatments were applied) were not significantly different (P > 0.20), and data were pooled for statistical purposes.
kg/pig daily, whereas RES pigs consumed 1.6 ± 0.2 kg/pig daily. There was a treatment × week interaction for BW gain (P < 0.01). As expected, the RES treatment successfully induced a decrease in the
29 ADG (Figure 1B). On average, FF pigs gained 0.90 ± 0.07 kg/d after wk 2 compared with 0.25 ± 0.06 kg/d for RES pigs. In addition, there was a treatment × week interaction for the G:F ratio (P < 0.01; Figure 1C). The G:F ratio for RES pigs was decreased on wk 1, 3, and 4 when compared with FF pigs. Feed restriction at 50, 40, and 20% of ad libitum intake was also associated with decreased ADG in pigs (Vandergrift et al., 1985; McNeel et al., 2000; Lovatto et al., 2006). Feed restriction decreases nutrient intake and consequently modifies metabolic activity and the utilization of energy. The G:F ratio of FF animals was comparable with that previously reported in control barrows that were fed a similar diet (Ji et al., 2006), except for the G:F ratio obtained on wk 5 in the present experiment. The significant decrease in the G:F ratio
RESULTS AND DISCUSSION Feed Intake and Body Weight Dietary manipulation was used in pigs to mimic extreme BW changes and to evaluate associated changes in ghrelin, GH, insulin, and glucose kinetics. A treatment × week interaction was associated with a decrease in feed intake by RES pigs beginning on wk 1 (P < 0.001; Figure 1A). On average, FF pigs consumed 3.7 ± 0.2
Figure 3. A) Insulin concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs (treatment × week interaction; P < 0.01). B) Insulin concentration in pigs before (fasting) and after (+1.5 h) feeding. Values are shown averaged over the data for FF and RES pigs. Asterisks (*) indicate significant differences in insulin for the given week (sampling time × week interaction; P < 0.01).
30
Barretero-Hernandez et al.
libitum for 5 wk), backfat thickness and adipocyte size was significantly decreased; however, no significant effects on the concentrations of anabolic transcripts associated with adipocytes were observed (McNeel et al., 2000). Taken together, it seems likely that the feed restriction in RES pigs signaled the GH neuroendocrine axis to increase GH concentrations.
Insulin and Glucose
Figure 4. A) Glucose concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs (P > 0.40). B) Glucose concentration in pigs before (fasting) and after (+1.5 h) feeding. Values are shown averaged over the data for FF and RES pigs. Asterisks (*) indicate significant differences in glucose for the given week (sampling time × week interaction; P < 0.01).
in RES animals can be attributed to the lower feed intake and decreased ingestion of amino acids (Veum et al., 1970; Chiba et al., 1991).
Growth Hormone There was a treatment × week interaction for GH concentrations (P < 0.01), which were increased for 4 consecutive weeks beginning on wk 2 of the experimental period in RES vs. FF pigs (Figure 2). No differences were observed in GH concentrations between fasting and 1.5 h after feeding (P > 0.20; data not shown). Growth hormone is secreted episodically in pigs (Clutter et al., 1995). In the present experiment, blood samples
were obtained at weekly intervals; therefore, an accurate estimation of pulsatile GH secretion was not feasible. Growth hormone is well known to play an important role in the regulation of metabolism, body composition, and energy expenditure. Undernutrition and dietary restriction induce a decrease in IGF-I and are associated with increased GH concentration in pigs (Kasser et al., 1981; Buonomo and Baile, 1991) and with GH resistance in most species (Hornick et al., 2000). It has been postulated that increased GH concentration during feed restriction allows fat mobilization to provide the additional energy needed to maintain homeostasis. When pigs were feed restricted (50% of ad
Insulin concentrations were increased in FF vs. RES pigs (treatment × week interaction, P < 0.01; Figure 3A); however, concentrations of glucose were not affected by treatment (Figure 4A). Given the significant differences in feed intake and the changes in insulin concentrations, reasons for the lack of response of glucose to treatment are not clear. Increased glucose concentration stimulates insulin release to facilitate uptake, utilization, and storage of glucose in muscle and adipose tissue (Kahn, 1996). Glucose transporters are activated when insulin binds to its plasma membrane receptors, allowing the cell to take up glucose and stimulate glycogen formation by activation of enzymatic systems. When glucose concentrations decrease, glucose transporters are recycled back into the cytoplasm, and glycogen synthesis stops (Shepherd and Kahn, 1999; Bryant et al., 2002). In the present experiment, insulin concentrations were increased in FF vs. RES pigs after 3 wk of treatment. Insulin promotes the uptake of fatty acids and the synthesis of lipids in the liver by increasing the availability of acetyl-CoA carboxylase and fatty acid synthase (Hillgartner et al., 1995). Changes in insulin, GH, and perhaps body fat mass in RES pigs may have resulted in changes in fatty acid metabolism. When lean and obese pigs were feed restricted (50% of ad libitum for 5 wk), backfat and fasting nonesterified fatty acid concentrations decreased in both lines (McNeel et al., 2000). In addition, fasting for 14 or 28 d significantly increased hepatic and renal gluconeogenesis and decreased
Feed restriction and metabolites in pigs
Figure 5. A) Ghrelin concentrations in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs (P > 0.20). B) Ghrelin concentration in pigs before (fasting) and after (+1.5 h) feeding. Values are shown averaged over the data for FF and RES pigs. There were significant sampling time and week effects on ghrelin concentrations (P < 0.01).
fatty acid synthesis and glucose oxidation in pigs (Klain et al., 1977). These data suggest that RES pigs had a lower lipolytic rate and enhanced generation of glucose from noncarbohydrate carbon substrates compared with FF pigs. Insulin and glucose concentrations were greater after feeding (+1.5 h) compared with concentrations after 18 h of fasting (sampling time × week interaction, P < 0.01; Figures 3B and 4B). This finding is consistent with the response observed in growing pigs, in which feed withdrawal for up to
48 h was associated with decreased insulin concentrations (Buonomo and Baile, 1991), reflecting the normal feedback mechanism devoted to maintain glucose homeostasis.
Ghrelin There were significant sampling time (P < 0.01) and week effects (P < 0.01) on ghrelin concentrations. On average, preprandial (fasting) ghrelin concentrations were 35.9 ± 0.9% greater than concentrations after feeding during the 7-wk experi-
31 mental period (Figure 5B). In gilts and weanling pigs, preprandial (72-h fasting) ghrelin concentrations were greater than those 6 to 48 h after feeding (Salfen et al., 2003; Govoni et al., 2005). Similar results have been reported in humans (Cummings et al., 2001), cattle (Hayashida et al., 2001), sheep (Sugino et al., 2004), and rodents (Tschop et al., 2000). In humans, plasma ghrelin concentrations rose almost 2-fold shortly before each meal and fell to base levels within 1 h after eating (Cummings et al., 2001). Additionally, administration of ghrelin to healthy human volunteers caused hunger sensations associated with increased plasma glucose followed by decreased insulin secretion (Broglio et al., 2001). Despite changes before and after feeding, concentrations of ghrelin were not significantly different (P > 0.20) between FF and RES pigs (Figure 5A). Systemic exogenous administration of ghrelin induces adiposity in rodents by stimulating an acute increase in feed intake, as well as a decrease in fat utilization (Tschop et al., 2000; Wren et al., 2000; Hayashida et al., 2001). However, as noted previously, the orexigenic effect of peripheral ghrelin seems to be more potent in young vs. adult rats (Gilg and Lutz, 2006), and mice that are null for the ghrelin receptor are subject to dietinduced obesity (Sun et al., 2008). In pigs, immunization against ghrelin is associated with decreased voluntary feed intake and decreased BW gain, providing evidence that ghrelin is involved in the long-term control of feed intake and BW in this species (Vizcarra et al., 2007). Peripheral infusion of ghrelin in 18-d-old pigs did not change feed intake; however, BW gain during the 5-d ghrelin infusion period was greater in treated piglets (Salfen et al., 2004). In the present study, ghrelin was responsive only to short-term feed withdrawal (18 h), but feed restriction for 5 wk was without effect. The lack of response of ghrelin to the 5-wk feed restriction program is perplexing because most of the published data suggest that ghrelin is involved in
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Table 1. Effect of feed restriction on glucose kinetics during a glucose tolerance test obtained on wk −1 and 5 in pigs that were allowed unrestricted access to feed and pigs that were fed 50% (BW basis) of the feed given to unrestricted pigs Week 5 Item
Week −11
FF2
G0 Clearance,5 %/min t1/2,6 min AUC7
373.4 1.78a 26.8a 5,166a
361.9 1.69a 26.9a 5,280a
4
a,b
a
RES3 a
SEM
359.9 2.47b 20.8b 3,150b a
14.5 0.17 1.5 604
Means within a row with different superscripts differ, P < 0.05.
1
Pooled data from all pigs before treatments were applied.
2
FF = full-fed pigs.
3
RES = pigs restricted to 50% (BW basis) of the feed provided to FF pigs.
4
G0 = peak glucose concentration.
5
Clearance rate per minute expressed as a percentage.
6
t1/2 = half-life.
7
AUC = area under the curve.
the long-term control of feed intake. Perhaps the length and degree of feed restriction imposed in the present experiment were not severe enough to produce a measurable effect on ghrelin secretion. Because only total ghrelin was measured, it is also possible that the variability observed in the present experiment is associated with differences between active and nonactive ghrelin forms. Further
research is needed to confirm and extend our results.
Glucose Tolerance Test All glucose kinetics obtained after the GTT procedures were significantly affected by treatment, except for peak glucose concentrations (Table 1). On wk 5, the glucose clearance rate was 62% greater in RES pigs than in FF animals. Consequently, the time
required for glucose concentrations to fall by half (t1/2) and the AUC were greater in FF than in RES pigs (Table 1; Figure 6). Insulin sensitivity was greater in RES animals compared with FF pigs on wk 5. Increased glucose clearance rate, decreased glucose t1/2, and decreased AUC during the GTT suggest that RES pigs were more responsive to endogenous insulin secretion in response to glucose infusion (Table 1; Figure 6). The glucose kinetics observed in FF pigs were similar to those reported in growing-finishing barrows consuming a diet similar to the one fed in the present experiment (Matthews et al., 2001). There is also good agreement of our data with those observed in humans. A decrease in BW resulting from restricted caloric intake or increased exercise improved glucose kinetics in obese patients (Gan et al., 2003; Weiss et al., 2006; Weiss and Holloszy, 2007). Taken together, our data suggest that the decreased tolerance for glucose in FF pigs on wk 5, as evidenced by longer t1/2 and greater AUC values compared with RES pigs, reflects the inability of the FF animals to dispose of a test dose of glucose with the same efficiency as RES pigs. In contrast, glucose kinetics in lactating sows were not affected by feed restriction (Quesnel et al., 2007). The differences between our results and those of Quesnel et al. (2007) probably reflect the degree of feed restriction, as well as differences in the physiological stage of the animals in the 2 studies. For example, glucose half-life is greater in pregnant sows than in nonpregnant animals (Pere et al., 2000). Nonetheless, the decrease of more than 6 min in glucose half-life in RES pigs at wk 5 can be deemed physiologically relevant because changes of the same extent were previously observed in pigs under contrasting physiological and nutritional treatments (Pere et al., 2000; Matthews et al., 2001).
IMPLICATIONS Figure 6. Average glucose concentrations during a glucose tolerance test (wk 5) in pigs that were allowed unrestricted access to feed (FF) and pigs that were fed 50% (BW basis; RES) of the feed given to FF pigs.
A significant preprandial increase in ghrelin was observed after fast-
Feed restriction and metabolites in pigs
ing; however, feed restriction for 5 consecutive weeks did not alter ghrelin concentrations, suggesting that plasma ghrelin concentrations are responsive only to short-term feed withdrawal. Alternatively, the length of time and severity of feed restriction imposed in the present experiment might not have been sufficient to produce measurable effects on ghrelin concentrations. Although ghrelin did not respond to feed restriction, feed-restricted pigs had increased GH and decreased insulin concentrations in plasma, and restricted pigs were more responsive to glucose clearance, half-life, and the AUC after glucose infusion than was the case with fullfed pigs.
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