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. 2019 Nov 4;97(12):4783–4791. doi: 10.1093/jas/skz336

Effects of central and peripheral administration of an acute-phase protein, α-1-acid-glycoprotein, on feed intake and rectal temperature in sheep

Brittany A Gregg 1,2, Paxton A Parker 1, Kathryn M Waller 1, Liesel G Schneider 2, Miriam Garcia 3, Barry Bradford 3, Joseph A Daniel 4, Brian K Whitlock 1,2,✉
PMCID: PMC6915238  PMID: 31679022

Abstract

In rodents, an acute-phase protein, α-1-acid-glycoprotein (AGP), was shown to provide a link between inflammation and suppression of feed intake by acting as a leptin receptor agonist. The objective of this study was to determine the effects of AGP on feed intake and rectal temperature in sheep. Ewes were ovariectomized, implanted with a cannula into a lateral ventricle of the brain, and kept indoors in individual pens. Feed intake and rectal temperature were determined for sheep in all experiments. In the first experiment, ewes (n = 4) received 1 of 4 treatments [0 (control), 0.012 (low), 0.06 (medium), or 0.30 (high) mg/kg BW AGP] into the lateral ventricle (ICV). All sheep received all treatments in a Latin square design balanced for carryover effects with 10 d between treatments. In the second experiment, ewes (n = 10) received 1 of 2 treatments (0 and 3 mg/kg BW of AGP) intravenously (IV) in a completely randomized design. In the third experiment, ewes (n = 19) received peripheral treatments (IV) of an antipyretic [0 (control) or 2.2 mg/kg BW flunixin meglumine (FLU)] 30 min before receiving central AGP [0 (control) or 0.3 mg/kg BW of AGP] in a completely randomized design. All data were analyzed using a mixed model analysis of variance and tested for effects of treatment, time, and the interaction of treatment and time. Cumulative 48-h feed intake after administration of treatments was also determined. In the first experiment, there was no effect of ICV treatment (P = 0.37) on feed intake rate or on cumulative feed intake (P = 0.31). There was an effect of ICV treatment (P = 0.002) on rectal temperatures, which were greater (P < 0.05) after the high dose of centrally administered AGP. In the second experiment, there was no effect of AGP administration IV on feed intake rate (P = 0.98), on cumulative feed intake (P = 0.41) or on rectal temperature (P = 0.71). In the third experiment, there was an effect of central AGP treatment (P < 0.0001) and an interaction of central AGP and time (P < 0.0001) on rectal temperature, whereas FLU had no effect (P = 0.93), demonstrating that AGP increased rectal temperatures regardless of antipyretic treatment. These results indicate that central AGP increases rectal temperature in sheep by pathways that do not involve prostaglandins. Further research is needed to determine whether AGP may be an important integrator of energy balance and inflammation.

Keywords: acute-phase protein, feed intake, orosomucoid, α-1-acid-glycoprotein, rectal temperature, sheep

Introduction

Inflammation and suboptimal feed intake are common in livestock during the transition from late gestation to lactation, and both conditions are associated with greater risk for removal from the dairy herd and less productivity (Bradford et al., 2015). Along with decreased feed intake, fever is also associated with inflammation (Ingvartsen and Andersen, 2000; Sartin et al., 2008).

Leptin, an adipokine synthesized and released into circulation in proportion to the amount of body fat, plays a crucial role in the regulation of food intake and energy expenditure (Blache et al., 2000; Delavaud et al., 2000; Ehrhardt et al., 2000). Administration of leptin reduces food intake and increases energy expenditure and body temperature (Halaas et al., 1995; Barb et al., 1998; Henry et al., 1999, 2008, 2011; Morrison et al., 2001; Skibicka and Grill, 2009). In rodents and primates, leptin gene expression in adipocytes and/or circulating concentrations of leptin are increased by bacterial endotoxin as well as proinflammatory cytokines such as tumor necrosis factor-α and IL-1β (Grunfeld et al., 1996; Finck et al., 1998; Francis et al., 1999; Landman et al., 2003). Therefore, leptin is an important hormone regulating food intake, metabolism, and body temperature in sick animals (Harden et al., 2006).

Although leptin regulates food intake (Chilliard et al., 2005) and endocrine events in ruminants (Morrison et al., 2001; Daniel et al., 2002; Zieba et al., 2005), plasma leptin in ruminants is unchanged by inflammation induced by endotoxin (lipopolysaccharide, LPS; Soliman et al., 2001, 2002; Daniel et al., 2002), leaving questions about the mechanisms by which inflammation and/or LPS induces changes in food intake, metabolism, and body temperature in domestic species such as sheep and cattle. One response of animals to natural and induced inflammation is the synthesis and secretion of acute-phase proteins into circulation. These proteins are generally produced by the liver, normally found in low abundance in the bloodstream, but are greatly elevated during periods of systemic inflammation (Murata et al., 2004; Petersen et al., 2004). Alpha-1-acid-glycoprotein (AGP) is an acute-phase protein with diverse roles. In rodents, AGP has anti-inflammatory and immunomodulatory properties (Hochepied et al., 2003). Bellinger and Mendel (1990) determined that central administration of AGP induced hypophagia in rodents. The putative role of AGP in appetite regulation was not evaluated further until a recent publication (Sun et al., 2016) thoroughly demonstrated the role of AGP as a hypophagic compound, signaling via the hypothalamic leptin receptor to reduce feed intake in rodents. Although these findings are interesting, it is exciting to consider the potential role of AGP as a link between inflammation, appetite, and temperature regulation in ruminants, as they lack the typical inflammation-induced leptin response observed in rodents and humans. Therefore, AGP may play an even more important role in the effects of inflammation on appetite and temperature regulation in ruminants than other species. The objectives of this study were to determine the effects of central and peripheral AGP administration on feed intake and rectal temperature in sheep and to help elucidate the mechanism(s) by which AGP increased rectal temperature in sheep.

Materials and Methods

The Institutional Animal Care and Use Committee of the University of Tennessee approved, in protocol number 2514-0217, all procedures and protocols used throughout this experiment.

Animals and Maintenance

Nonlactating, nonpregnant mature (≥1 yr of age) Suffolk or Suffolk-cross female sheep were ovariectomized to avoid cyclic variations in plasma concentrations of steroids (as this may affect feed intake; Laker et al., 2011) and allowed at least 2 wk for recovery before subsequent surgeries and at least 1 mo before use in any experiments. Each sheep was then fitted with an intracerebroventricular (ICV) catheter into a lateral ventricle of the brain as previously described (Whitlock et al., 2010). Twice each week while the ICV catheter remained in place, antibiotics were administered to prevent potential infections and help maintain catheter patency. Catheter patency was assessed each time antibiotics were administered centrally by attempting to withdraw approximately 1 mL of cerebrospinal fluid (CSF) from the subcutaneous port and ICV catheter unit. The ICV antibiotic “cocktail” included Gentamycin (Hospira, Inc., Lake Forest, IL) and Vancomycin (Hospira, Inc.). Approximately 100 µL of the antibiotic cocktail [Gentamycin (5 mg/mL) and Vancomycin (10 mg/mL)] was administered through the port and ICV catheter and subsequently flushed with approximately 250 µL of sterile, nonpyrogenic, isotonic, and 0.9% sodium chloride (Hospira, Inc.). Based on previous experience, at least 80% of the ICV catheters and subcutaneous ports were expected to remain patent for subsequent experimental use; therefore, additional animals were prepared in anticipation of a failure rate of no more than 20%. For all ICV injections, the skin above the port site was aseptically prepared with 70% isopropyl alcohol before all central treatments. Treatments were administered through the skin and into the port via a 25-gauge Huber Point needle (Norfolk Vet Products, Skokie, IL) followed with 250 µl of sterile, nonpyrogenic, isotonic, 0.9% sodium chloride to flush the port and catheter. Ewes were kept indoors in individual pens (approximately 3 m2) with an environment consisting of a 12-h light/dark photoperiod at approximately 22 to 24 °C. Ewes were fed a diet calculated to meet 100% of daily maintenance requirements (National Research Council, 2007) and had ad libitum access to water.

Experiment 1—Effects of Central Administration of α-1-Acid-Glycoprotein on Feed Intake and Rectal Temperature in Sheep

Ovariectomized sheep (n = 4) weighing 79.0 ± 5.0 (SD) kg, with patent ICV catheters and ports, received 1 of 4 treatments [0 (control), 0.012 (low), 0.060 (medium), or 0.30 (high) mg/kg BW AGP (from bovine plasma; Sigma Aldrich Co., Saint Louis, MO)] administered in 500 µL of sterile, nonpyrogenic, isotonic, and 0.9% sodium chloride into the lateral ventricle. The day before each experimental period BW were determined. A 10-d interval was allowed between experimental periods and each period lasted 5 d, with 3 d for basal measure of feed intake and rectal temperature and 2 d for measurement of feed intake and rectal temperature after experimental treatment administration. Experimental treatments were administered as a single bolus reconstituted in 500 μL of sterile, nonpyrogenic, isotonic, and 0.9% sodium chloride (Daniel et al., 2016). Feed intake and rectal temperature were determined at −72, −48, −24, 0, 2, 4, 6, 8, 12, 24, 36, and 48 h relative to treatment (time 0: administration of the respective treatments). Feed intake was determined by weighing feed not consumed, calculating feed intake, and replenishing consumed feed at each time point. At no time were sheep without feed. All sheep received all treatments in random order a Latin square design balanced for carryover effects with 10 d between treatments.

Experiment 2—Effects of Peripheral Administration of α-1-Acid-Glycoprotein on Feed Intake and Rectal Temperature in Sheep

Ewes (n = 10) weighing 78.5 ± 23.7 (SD) kg were blocked by BW and then randomly assigned within block to 2 IV treatments (0 and 3.0 mg/kg BW of AGP). Approximately 24 h before treatment administration, sheep BW were determined and the area over each animals’ jugular vein was desensitized by SC administration of 2% lidocaine hydrochloride solution (VetOne, Meridian, ID) before inserting a catheter (16-gauge, 13-cm, Extended Use MILACATH catheter, Mila International Inc., Florence, KY) into the vein. The catheter was attached to an extension set (18-cm MicroCLAVE Smallbore T-Connector, Abbott Laboratories, North Chicago, IL) and then sutured to the ewe’s neck. The catheters and extension sets were flushed with heparinized saline (0.9% sodium chloride) solution (20 U of heparin/mL). Each ewe was infused via IV catheters with 1 of 2 doses [0 (control; n = 5) or 3.0 (AGP; n = 5) mg/kg BW] of a commercially available AGP extracted from bovine plasma. α-1-Acid-glycoprotein was approximately 10-fold greater than the greatest dose administered centrally in Exp. 1 (0.30 mg/kg BW). Using a 10-fold greater dose has been utilized in previous experiments when going from central to peripheral treatment administration (Whitlock et al., 2010). AGP was administered as a single bolus reconstituted in approximately 1 mL of pyrogen-free saline (Daniel et al., 2016). The sheep were offered a known amount of fresh feed ad libitum. Feed was weighed and replaced with fresh feed, and rectal temperatures were determined at −72, −48, −24, 0, 2, 4, 6, 8, 12, 24, 36, and 48 h (time 0: administration of the respective treatments). Feed intake was determined as described for Exp. 1.

After a washout period of ≥10 d, sheep were included in subsequent experimentations (Exp. 3).

Experiment 3—Effects of Peripheral Administration of a Nonsteroidal Anti-inflammatory, Flunixin Meglumine, and Central Administration of α-1-Acid-Glycoprotein on Feed Intake and Rectal Temperature in Sheep

Ewes (n = 19; weighing 85.4 ± 19.4 kg) previously ovariectomized and implanted with an ICV catheter and port were used in this experiment. Approximately 24 h before treatment administration, BW were determined and jugular catheters were inserted and maintained as described in Experiment 2. Sheep were blocked by BW and then randomly assigned within block to receive peripheral treatments [administered IV; 0 (SAL; equal volume of saline to match the other IV treatment) or 2.2 mg/kg BW flunixin meglumine (FLU {Prevail; VetOne}; a cyclo-oxygenase inhibitory nonsteroidal anti-inflammatory drug)] 30 min before receiving centrally [administered ICV through a port and catheter into the lateral ventricle of the brain; 500 µL of SAL or 25 mg AGP (approximately 0.3 µg/kg BW) administered in 500 µL of sterile, nonpyrogenic, isotonic, 0.9% sodium chloride] to yield four possible treatment combinations (SAL/SAL, n = 4; FLU/SAL, n = 5; SAL/AGP, n = 5; FLU/AGP, n = 5). Feed intake and rectal temperature were determined at −48, −24, −2, 0, 2, 4, 6, 8, 12, and 24 h relative to AGP treatment (time 0: administration of the AGP treatments). Feed intake was determined as described for Experiment 1.

Statistical Analysis

To determine the effects on feed intake rate (kg of feed intake/time), cumulative feed intake, and rectal temperature throughout the experiments, data were analyzed using the GLIMMIX procedure for mixed model analysis of variance (SAS software, version 9.4 SAS Institute Inc., Cary, NC). Values of P < 0.05 were considered significant, and values of P ≥ 0.05 to P ≤ 0.10 were considered a tendency.

To determine the effects on feed intake rate, cumulative feed intake, and rectal temperature in Exp. 1, data were analyzed using a 4 × 4 Latin square design. Data for feed intake rate and rectal temperature were tested for fixed effects of treatment, time, and their interactions and the random effects of sheep and period. Repeated measures of time were accounted for with feed intake rate and rectal temperature. Cumulative feed intake after administration of treatments was tested for fixed effect of treatment and the random effects of sheep and period.

To determine the effects on feed intake rate, cumulative feed intake, and rectal temperature in Exp. 2, data were analyzed as a completely randomized design. Data for feed intake rate and rectal temperature were tested for fixed effects of treatment, time, and their interactions and the random effects of sheep within treatments. Repeated measures of time were accounted for with feed intake rate and rectal temperature. Cumulative feed intake after administration of treatments was tested for the fixed effect of treatment.

To determine the effects on feed intake rate, cumulative feed intake, and rectal temperature in Exp. 3, data were analyzed as a completely randomized design with factorial treatment arrangement. Data for feed intake rate and rectal temperature were tested for fixed effects of central treatment, peripheral treatment, time, and their interactions and the random effect of sheep within treatment. Repeated measures of time were accounted for with feed intake rate and rectal temperature. Cumulative feed intake after administration of treatments was tested for fixed effects of central treatment, peripheral treatment, and their interaction.

Results

Experiment 1

Feed intake and rectal temperature did not differ during the pretreatment period (data not shown). There was an effect of time (P < 0.0001), but no effect of central AGP treatment (P = 0.37), or treatment by time interaction (P = 0.97) on feed intake rate (Fig. 1A), and there was no effect of treatment (P = 0.31) on 48-h cumulative feed intake (Fig. 1B). There was a tendency for an interaction of treatment and time (P = 0.07), and there was an effect of time (P < 0.0001) and AGP treatment (P = 0.002) on rectal temperature (Fig. 1C). Rectal temperatures were greater (P < 0.05) with the high dose compared with other AGP doses given.

Figure 1.

Figure 1.

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Effects of central α-1-acid-glycoprotein (AGP) administration on (A) feed intake rate (kg/h), (B) cumulative feed intake (kg), and (C) rectal temperatures (°C) in sheep (± SEM). Ewes were treated with 1 of 4 treatments [0 (control; n = 4), 0.012 (low; n = 4), 0.060 (medium; n = 4), or 0.30 (high; n = 4) mg/kg BW AGP] administered in 500 µL of sterile, nonpyrogenic, and 0.9% sodium chloride into the lateral ventricle at time 0 h. All sheep received all treatments in a Latin square design balanced for carryover effects with 10 d between treatments. (A) There was an effect of time (P < 0.0001) and no effect of treatment (P = 0.37), or treatment × time interaction (P = 0.97) on feed intake rate. (B) There was no effect of treatment (P = 0.31) on 48-h cumulative feed intake. (C) There was a tendency for an interaction of treatment and time (P = 0.07), and there was an effect of time (P < 0.0001) and treatment (P = 0.002) on rectal temperature. Rectal temperatures were greater (P < 0.05) with high dose compared with other doses given.

Experiment 2

Feed intake and rectal temperature did not differ during the pretreatment period (data not shown). There was no effect of IV AGP treatment (P = 0.98) or treatment by time interaction (P = 0.77), but there was an effect of time (P = 0.004) on feed intake rate (Fig. 2A). There was no effect of IV treatment (P = 0.41) on 48-h cumulative feed intake (Fig. 2B). There was a tendency for an effect of time (P = 0.10), but no effect of treatment (P = 0.71) or treatment by time interaction (P = 0.91) on rectal temperature (Fig. 2C).

Figure 2.

Figure 2.

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Effects of peripheral α-1-acid-glycoprotein (AGP) administration on (A) feed intake rate (kg/h), (B) cumulative feed intake (kg), and (C) rectal temperatures (°C) in sheep (± SEM). Ewes were treated with 1 of 2 treatments [0 (control; n = 5) and 3.0 (AGP; n = 5) mg/kg BW of AGP] infused via IV catheters at time 0 h to yield 2 possible treatment groups. The dose of AGP was selected based on the greatest dose administered centrally in Exp. 1 (0.30 mg/kg BW). (A) There was no effect of IV treatment (P = 0.98) or treatment × time interaction (P = 0.77), but there was an effect of time (P = 0.004) on feed intake rate. (B) There was no effect of IV treatment (P = 0.41) on 48-h cumulative feed intake. (C) There was a tendency for an effect of time (P = 0.10), but no effect of treatment (P = 0.71) or treatment × time interaction (P = 0.91) on rectal temperature.

Experiment 3

Feed intake and rectal temperature did not differ during the pretreatment period (data not shown). There was no effect of central AGP treatment administration (P = 0.18), time (P = 0.28), or AGP by time interaction (P = 0.64) on feed intake rate (Fig. 3A). Moreover, there was no effect of intravenous FLU treatment (P = 0.88), FLU by time (P = 0.21), FLU by AGP (P = 0.42), or FLU by AGP by time interactions (P = 0.50) on feed intake rate (Fig. 3A). There was no effect of central AGP treatment (P = 0.79), intravenous FLU treatment (P = 0.92), or their interaction (P = 0.32) on 24-h cumulative feed intake (Fig. 3B). There was an effect of central AGP administration (P < 0.0001), time (P < 0.0001), and AGP by time interaction (P < 0.0001) on rectal temperature such that ewes treated centrally with AGP had greater rectal temperatures at 2, 4, 6, 8, and 12 h relative to ewes treated centrally with saline (Fig. 3C). There was no effect of intravenous FLU treatment (P = 0.93), FLU by time (P = 0.35), FLU by AGP (P = 0.54), or FLU by AGP by time interactions (P = 0.15) on rectal temperature (Fig. 3C).

Figure 3.

Figure 3.

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Effects of central α-1-acid-glycoprotein (AGP) and peripheral flunixin meglumine administration on (A) feed intake rate (kg/h), (B) cumulative feed intake (kg), and (C) on rectal temperatures (°C) in sheep (± SEM). Ewes were treated with peripheral treatments [administered IV; 0 (SAL) or 2.2 mg/kg BW flunixin meglumine (FLU)] 30 min before receiving centrally [administered ICV through a port and catheter into the lateral ventricle of the brain; 0 (control; SAL) or 25 (AGP) mg AGP (approximately 0.30 mg/kg BW) to yield four possible treatment combinations (SAL/SAL, n = 4; FLU/SAL, n = 5; SAL/AGP, n = 5; FLU/AGP, n = 5). (A) There was no effect of central treatment administration (P = 0.18), time (P = 0.28), or central treatment by time interaction (P = 0.64) on feed intake rate. Moreover, there was no effect of intravenous treatment (P = 0.88), intravenous treatment by time interaction (P = 0.21), intravenous by central treatment (P = 0.42), or intravenous by central treatment by time interaction (P = 0.50) on feed intake rate. (B) There was no effect of central treatment (P = 0.79), intravenous treatment (P = 0.92), or their interaction (P = 0.32) on 24-h cumulative feed intake. (C) There was an effect of central treatment administration (P < 0.0001), time (P < 0.0001), and central treatment by time interaction (P < 0.0001) on rectal temperature such that ewes treated centrally with AGP had greater rectal temperatures at 2, 4, 6, 8, and 12 h relative to ewes treated centrally with SAL. There was no effect of intravenous treatment (P = 0.93), intravenous treatment by time interaction (P = 0.35), intravenous by central treatment (P = 0.54), or intravenous by central treatment by time interaction (P = 0.15) on rectal temperature. *Time points at which central AGP-treated ewes differed from central SAL-treated ewes (P < 0.05).

Discussion

Identifying the direct causes of intake depression is critical to preventing a cascade of disorders in animals undergoing exaggerated systemic inflammation. Sun et al. (2016) clearly demonstrated in rodents that AGP could potentially provide a link between inflammation and reduced feed intake via the leptin receptor. Our results show that AGP can induce an increase in rectal temperature in the ruminant model, sheep. Our discovery of a rectal temperature increase was found to be independent of the prostaglandin pathway. Feed intake was not affected by AGP administration.

Feed Intake

It is well documented that during the transition period, dairy cows undergo a marked systemic inflammation accompanied by an array of responses, some of which may be adaptive and others pathological. One of the most dramatic responses putatively tied to exaggerated peripartum inflammation is suppressed feed intake. Cairoli et al. (2006) demonstrated that AGP is elevated in early lactation in cattle, and a recent study confirmed that AGP has potent hypophagic effects in mice (Sun et al., 2016). Interestingly, although AGP suppressed food intake in ob/ob mice, it had no effect on feed intake in db/db mice (Sun et al., 2016), leading the authors to speculate that the effects of AGP on feed intake were mediated through the leptin receptor (LepR). Sun et al. (2016) confirmed their speculation when administration of AGP failed to affect feed intake in mice with temporarily silenced LepR, and they ultimately demonstrated with rat hypothalamic tissue and GT1-7 cells that AGP bound to and activated the LepR.

In our first study, the effect of AGP on feed intake was not significant. The conflicting findings relative to responses in mice could be because the central route of AGP administration has a lesser or no effect on feed intake, as the reported responses in mice were after IV or intraperitoneal AGP administration (Sun et al., 2016). However, it was observed previously that central administration of AGP decreased feed intake in rats (Bellinger and Mendel, 1990). While studying the effects of satietin on feed intake, Bellinger and Mendel inadvertently administered AGP. Ultimately, they determined that the satietin that they were administering also contained albumin and AGP; when AGP alone was administered centrally to rats, feed intake decreased (Bellinger and Mendel, 1990). Therefore, it is unlikely that the route by which we administered AGP in this first experiment was the reason feed intake was not significantly decreased.

The effects of AGP on feed intake in mice reported by Sun et al. (2016) were observed after peripheral administration (intraperitoneal and IV). Thus, we administered AGP peripherally in our second experiment. The dose of AGP used in the second experiment (~3.0 mg/kg IV; ~250 mg) was 10-fold greater than the greatest dose used in our first experiment (~0.30 mg/kg ICV; ~25 mg) to address greater circulating concentrations of acute-phase proteins and the likely limits to central nervous system access. However, similar to the first experiment, AGP had no effect on feed intake in sheep (cumulative or rate). Sun et al. (2016) administered 100 mg/kg IV or ~2 mg total AGP to reduce feed intake in mice. Based on their reported body weights, assumed blood volumes, and previously reported circulating concentrations of AGP in mice (Kopf et al., 1994), it is likely that the 2 mg total dose of AGP administered to the mice increased plasma AGP by approximately 670%. In cattle, serum concentrations of AGP vary. Serum concentrations of AGP range from approximately 200 to 450 mg/L in healthy cattle and increase to ≥1,000 mg/L during acute inflammation (Ceciliani et al., 2012). Recently, we observed a negative association between feed intake and serum AGP concentrations in periparturient dairy cattle (Brown et al., 2019). In that study, a reduction in periparturient intake of feed in dairy cattle was associated with an increase of approximately 40% in plasma AGP. Although this association does not confirm causation, it is interesting to consider in light of what was previously observed and reported regarding AGP and feed intake by Sun et al. (2016).

It is also possible that the lack of an effect of centrally administered AGP on feed intake in sheep was because the maximum dose given was not sufficient to elicit a hypophagic effect. When compared to the dose of AGP administered centrally to rats (50 μg, or approximately 0.20 mg/kg BW; Bellinger and Mendel, 1990), the maximum dose used in Exp. 1 and the dose in Exp. 3 of this study were greater (approximately 25 mg or approximately 0.30 mg/kg BW). Therefore, the difference in response as it relates to feed intake between rats and sheep after central administration of AGP cannot simply be explained by differences in doses used. That being stated, it is still possible that a greater central dose of AGP is needed to suppress/reduce feed intake in ruminants. If AGP truly has a role in inflammation-induced suppression of feed intake in sheep, maybe the concentrations (in plasma and possibly in CSF) achieved during systemic inflammation in this species, and necessary to affect feed intake, exceed that administered in our model. Unfortunately, we have been unable to find an existing assay allowing for accurate determination of serum AGP concentration in sheep samples.

Although there are very few reported reference ranges for serum concentrations of AGP in sheep, a 30-d experiment was conducted with the concentrations of the control sheep being approximately 100 mg/L (Eckersall et al., 2007). Concentrations in other species (mice, rats, and swine) are similar to those observed/reported in cattle (Poüs et al., 1990; Caperna et al., 2017). Therefore, it is likely that serum concentrations of AGP in healthy sheep are not that divergent from other species. Comparatively, if we assume that the normal circulating concentration of AGP in sheep is approximately 300 mg/L, the IV dose administered to the sheep in this study likely increased their serum AGP by 10% to 13%. If AGP does suppress feed intake in ruminants during inflammation, it is possible that the IV dose of AGP administered to our sheep was not comparable to increases in serum or plasma normally achieved during systemic inflammation associated with suppressed feed intake. Therefore, it is possible that a greater IV dose could have an effect on feed intake in sheep.

Bovine AGP includes at least five glycosylation sites and potentially eight phosphorylation sites (Ceciliani et al., 2012). Moreover, it is clear that systemic inflammation alters AGP glycosylation (Kreisman and Cobb, 2012). Therefore, it is possible that there are unique AGP glycosylations associated with inflammation that make the AGP molecule respond differently at the site of the LepR. Because of the complexity of this protein and changes associated with inflammation, we cannot rule out that the observations made during our studies resulted from differences in glycosylation of the AGP administered to our animals compared with endogenous AGP in sheep with inflammation. We did use the same lot of AGP for all studies reported herein, limiting concerns about variability of glycosylation status across animals.

Rectal Temperature

A previously unreported effect of central administration of AGP was our observation that it increased rectal temperature in sheep. Although this was a surprising and interesting observation, the mechanism(s) by which AGP increased rectal temperature in sheep remains unclear. Fever, an elevation in body temperature, may be the result of many infectious or inflammatory diseases. To elicit a fever, the infection/inflammatory stimulus triggers production of cytokines, which then use specific routes crossing the blood–brain barrier to initiate a febrile response. A rise in body temperature is necessary in many cases for the body to overcome an infection (for more in-depth review, see Evans et al., 2015).

One potential concern regarding the increased rectal temperature in response to central AGP administration was that the administered bovine AGP might have been contaminated with endotoxin. However, we determined that our AGP did not contain endotoxin using a limulus amebocyte lysate test. Moreover, the timing of the increased rectal temperature after central administration of AGP was not consistent with previous endotoxin models, in that the increase in temperature was later than what would have been observed after endotoxin administration (Kabaroff et al., 2006; Feng et al., 2010; Ranjan et al., 2011). Once we confirmed that the AGP did not contain endotoxin we turned our attention to other possible mechanisms.

It has been determined that central leptin administration in rats not only reduced feed intake but also increased body temperature (Luheshi et al., 1999; Turek et al., 2004; Skibicka and Grill, 2009). If the effect of AGP to suppress feed intake in rodents is mediated through the LepR as supported by Sun et al. (2016), then it is reasonable to speculate that AGP, like leptin, could increase body temperature. It could be that the effects of AGP or leptin on body temperature in rodents and/or sheep are more sensitive than the effects of either of them on feed intake. However, Luheshi et al. (1999) administered various quantities of leptin centrally to rats and found that even their lowest dose of 0.4 µg per rat caused a significant decrease in food intake, but did not increase temperature. Assuming greater doses of leptin are required to increase body temperature vs. suppress feed intake, it is reasonable to expect that AGP signaling via the LepR might act similarly. Still, we cannot rule out possible differences between species or between LepR ligands. It has also been stated that leptin-induced increase in body temperature in rats will plateau even when given up to 10 times greater than the normal plasma concentrations of leptin (Luheshi et al., 1999). Therefore, it is plausible to assume, if the temperature response was maximal in this study, that greater amounts of AGP may decrease feed intake in sheep while not causing any greater increase in rectal temperature. However, ultimately we concluded that we should first administer AGP by a different route to allow for more direct comparisons with previous work in mice.

Unlike central administration, peripheral administration of AGP had no effect on rectal temperatures. The peripheral dose of AGP needed to increase rectal temperature may not have been achieved. When administering leptin peripherally in rodents, the dose that caused an increase in temperature was 1 mg per rat (~3.5 mg/kg), although the increase in temperature was less than that induced by central leptin administration (Luheshi et al., 1999). The peripheral dose required to match responses to central administration may be more than the 10-fold increase that we used.

In one report, while studying the effects of leptin on feed intake and body temperature in rats, Luheshi et al. (1999) administered a cyclo-oxygenase inhibitor, flurbiprofen, and was able to block the leptin-induced increased temperature. Therefore, to help elucidate the mechanism(s) by which central administration of AGP increases rectal temperature in sheep, we designed a similar experiment to eliminate pyrogenic pathways that involve prostaglandins. We administered flunixin meglumine, a nonsteroidal anti-inflammatory drug and potent cyclo-oxygenase inhibitor that has antipyrogenic effects involving inhibition of endogenous pyrogens (IL-1 and tumor necrosis factor-α) and prostaglandin PGE2, at a dose labeled for ruminants for the control of pyrexia and inflammation associated with endotoxemia (McKellar et al., 1989; Lees and Taylor, 1991). Pretreatment with flunixin meglumine had no effect on increased rectal temperature after central administration of AGP. We also found no effect on feed intake. We concluded that central AGP administration increases rectal temperature in sheep by a mechanism(s) independent of prostaglandin synthesis.

However, more recent research indicates a prostaglandin-independent mechanism for leptin-induced increased rectal temperature in rodents (Turek et al., 2004; Skibicka and Grill, 2009). In those experiments, the leptin-induced increase in rectal temperature was affected by agonists and antagonists of downstream leptin effects on hypothalamic neuropathways, specifically melanocortin receptors (Turek et al., 2004; Skibicka and Grill, 2009). Therefore, it is plausible that central administration of AGP has an effect on body tsemperature/rectal temperature through actions at the LepR and subsequently hypothalamic melanocortin receptors. Subsequent studies designed to determine the mechanism(s) by which AGP increases rectal temperature in sheep might focus on the effects of blocking hypothalamic melanocortin receptors before central administration of AGP.

Conclusions

Although AGP did not decrease feed intake in our sheep model, central administration of AGP did increase rectal temperature. Peripheral administration of 10 times the central dose failed to induce measurable responses in either feed intake or body temperature. Furthermore, administration of flunixin meglumine did not abolish the increase rectal temperature brought on by central administration of AGP, making it unlikely that prostaglandins mediate AGP-stimulated increases in rectal temperature. α-1-Acid-glycoprotein may be an important integrator of inflammation, food intake, and body temperature in ruminants; however, further research is needed to completely elucidate its roles in these areas.

Acknowledgments

This work was supported by U.S. Department of Agriculture | National Institute of Food Agriculture (017-67015-26491). Any opinions, findings, and conclusions or recommendations expressed in this manuscript are those of the authors and do not necessarily reflect the views of the funding agencies. The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Literature Cited

  1. Barb C. R., Yan X., Azain M. J., Kraeling R. R., Rampacek G. B., and Ramsay T. G.. . 1998. Recombinant porcine leptin reduces feed intake and stimulates growth hormone secretion in swine. Domest. Anim. Endocrinol. 15:77–86. [DOI] [PubMed] [Google Scholar]
  2. Bellinger L. L., and Mendel V. E.. . 1990. The effects of semi- and HPLC-purified human satietin and alpha-1-glycoprotein on ingestion and body weight. Brain Res. Bull. 25:941–947. doi: 10.1016/0361-9230(90)90192-3 [DOI] [PubMed] [Google Scholar]
  3. Blache D., Tellam R. L., Chagas L. M., Blackberry M. A., Vercoe P. E., and Martin G. B.. . 2000. Level of nutrition affects leptin concentrations in plasma and cerebrospinal fluid in sheep. J. Endocrinol. 165:625–637. doi: 10.1677/joe.0.1650625 [DOI] [PubMed] [Google Scholar]
  4. Bradford B. J., Yuan K., Farney J. K., Mamedova L. K., and Carpenter A. J.. . 2015. Invited review: Inflammation during the transition to lactation: New adventures with an old flame. J. Dairy Sci. 98:6631–6650. doi: 10.3168/jds.2015-9683 [DOI] [PubMed] [Google Scholar]
  5. Brown W. E., Garcia M., Mamedova L. K., Zenobi M. G., Staples C. R., Leno B. M., Overton T. R., Whitlock B. K., Daniel J. A., and Bradford B. J.. . 2019. Plasma alpha-1-acid glycoprotein is negatively associated with dry matter intake in postpartum dairy cows. J. Dairy Sci. 102(Suppl. 1):395 (Abstr.) [DOI] [PubMed] [Google Scholar]
  6. Cairoli F., Battocchio M., Veronesi M. C., Brambilla D., Conserva F., Eberini I., Wait R., and Gianazza E.. . 2006. Serum protein pattern during cow pregnancy: Acute-phase proteins increase in the peripartum period. Electrophoresis 27:1617–1625. doi: 10.1002/elps.200500742 [DOI] [PubMed] [Google Scholar]
  7. Caperna T. J., Shannon A. E., Stoll M., Kahl S., Blomberg L. A., Vallet J. L., and Ramsay T. G.. . 2017. A sandwich ELISA for porcine alpha-1 acid glycoprotein (pAGP, ORM-1) and further demonstration of its use to evaluate growth potential in newborn pigs. Domest. Anim. Endocrinol. 60:75–82. doi: 10.1016/j.domaniend.2017.04.001 [DOI] [PubMed] [Google Scholar]
  8. Ceciliani F., Ceron J. J., Eckersall P. D., and Sauerwein H.. . 2012. Acute phase proteins in ruminants. J. Proteomics 75:4207–4231. doi: 10.1016/j.jprot.2012.04.004 [DOI] [PubMed] [Google Scholar]
  9. Chilliard Y., Delavaud C., and Bonnet M.. . 2005. Leptin expression in ruminants: Nutritional and physiological regulations in relation with energy metabolism. Domest. Anim. Endocrinol. 29:3–22. doi: 10.1016/j.domaniend.2005.02.026 [DOI] [PubMed] [Google Scholar]
  10. Daniel J. A., Whitlock B. K., Baker J. A., Steele B., Morrison C. D., Keisler D. H., and Sartin J. L.. . 2002. Effect of body fat mass and nutritional status on 24-hour leptin profiles in ewes. J. Anim. Sci. 80:1083–1089. doi: 10.2527/2002.8041083x [DOI] [PubMed] [Google Scholar]
  11. Daniel J. A., Whitlock B. K., Marks D. L., Gard J. A., and Sartin J. L.. . 2016. Leukemia inhibitory factor as a mediator of lipopolysaccharide effects on appetite and selected hormones and metabolites. J. Anim. Sci. 94:2789–2797. doi: 10.2527/jas.2016-0396 [DOI] [PubMed] [Google Scholar]
  12. Delavaud C., Bocquier F., Chilliard Y., Keisler D. H., Gertler A., and Kann G.. . 2000. Plasma leptin determination in ruminants: Effect of nutritional status and body fatness on plasma leptin concentration assessed by a specific RIA in sheep. J. Endocrinol. 165:519–526. doi: 10.1677/joe.0.1650519 [DOI] [PubMed] [Google Scholar]
  13. Eckersall P. D., Lawson F. P., Bence L., Waterston M. M., Lang T. L., Donachie W., and Fontaine M. C.. . 2007. Acute phase protein response in an experimental model of ovine caseous lymphadenitis. BMC Vet. Res. 3:35. doi: 10.1186/1746-6148-3-35 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ehrhardt R. A., Slepetis R. M., Siegal-Willott J., Van Amburgh M. E., Bell A. W., and Boisclair Y. R.. . 2000. Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep. J. Endocrinol. 166:519–528. doi: 10.1677/joe.0.1660519 [DOI] [PubMed] [Google Scholar]
  15. Evans S. S., Repasky E. A., and Fisher D. T.. . 2015. Fever and the thermal regulation of immunity: The immune system feels the heat. Nat. Rev. Immunol. 15:335–349. doi: 10.1038/nri3843 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feng S. Y., Samarasinghe T., Phillips D. J., Alexiou T., Hollis J. H., Yu V. Y., and Walker A. M.. . 2010. Acute and chronic effects of endotoxin on cerebral circulation in lambs. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298:R760–R766. doi: 10.1152/ajpregu.00398.2009 [DOI] [PubMed] [Google Scholar]
  17. Finck B. N., Kelley K. W., Dantzer R., and Johnson R. W.. . 1998. In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide. Endocrinology 139:2278–2283. doi: 10.1210/endo.139.5.6012 [DOI] [PubMed] [Google Scholar]
  18. Francis J., MohanKumar P. S., MohanKumar S. M., and Quadri S. K.. . 1999. Systemic administration of lipopolysaccharide increases plasma leptin levels: Blockade by soluble interleukin-1 receptor. Endocrine 10:291–295. doi: 10.1007/BF02738628 [DOI] [PubMed] [Google Scholar]
  19. Grunfeld C., Zhao C., Fuller J., Pollack A., Moser A., Friedman J., and Feingold K. R.. . 1996. Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. J. Clin. Invest. 97:2152–2157. doi: 10.1172/JCI118653 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Halaas J. L., Gajiwala K. S., Maffei M., Cohen S. L., Chait B. T., Rabinowitz D., Lallone R. L., Burley S. K., and Friedman J. M.. . 1995. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546. doi: 10.1126/science.7624777 [DOI] [PubMed] [Google Scholar]
  21. Harden L. M., du Plessis I., Poole S., and Laburn H. P.. . 2006. Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior. Physiol. Behav. 89:146–155. doi: 10.1016/j.physbeh.2006.05.016 [DOI] [PubMed] [Google Scholar]
  22. Henry B. A., Andrews Z. B., Rao A., and Clarke I. J.. . 2011. Central leptin activates mitochondrial function and increases heat production in skeletal muscle. Endocrinology 152:2609–2618. doi: 10.1210/en.2011-0143 [DOI] [PubMed] [Google Scholar]
  23. Henry B. A., Dunshea F. R., Gould M., and Clarke I. J.. . 2008. Profiling postprandial thermogenesis in muscle and fat of sheep and the central effect of leptin administration. Endocrinology 149:2019–2026. doi: 10.1210/en.2007-1311 [DOI] [PubMed] [Google Scholar]
  24. Henry B. A., Goding J. W., Alexander W. S., Tilbrook A. J., Canny B. J., Dunshea F., Rao A., Mansell A., and Clarke I. J.. . 1999. Central administration of leptin to ovariectomized ewes inhibits food intake without affecting the secretion of hormones from the pituitary gland: Evidence for a dissociation of effects on appetite and neuroendocrine function. Endocrinology 140:1175–1182. doi: 10.1210/endo.140.3.6604 [DOI] [PubMed] [Google Scholar]
  25. Hochepied T., Berger F. G., Baumann H., and Libert C.. . 2003. Alpha(1)-acid glycoprotein: An acute phase protein with inflammatory and immunomodulating properties. Cytokine Growth Factor Rev. 14:25–34. [DOI] [PubMed] [Google Scholar]
  26. Ingvartsen K. L., and Andersen J. B.. . 2000. Integration of metabolism and intake regulation: A review focusing on periparturient animals. J. Dairy Sci. 83:1573–1597. doi: 10.3168/jds.S0022-0302(00)75029-6. [DOI] [PubMed] [Google Scholar]
  27. Kabaroff L. C., Rodriguez A., Quinton M., Boermans H., and Karrow N. A.. . 2006. Assessment of the ovine acute phase response and hepatic gene expression in response to Escherichia coli endotoxin. Vet. Immunol. Immunopathol. 113:113–124. doi: 10.1016/j.vetimm.2006.04.003. [DOI] [PubMed] [Google Scholar]
  28. Kopf M., Baumann H., Freer G., Freudenberg M., Lamers M., Kishimoto T., Zinkernagel R., Bluethmann H., and Köhler G.. . 1994. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368:339–342. doi: 10.1038/368339a0 [DOI] [PubMed] [Google Scholar]
  29. Kreisman L. S. C., and Cobb B. A.. . 2012. Infection, inflammation and host carbohydrates: A Glyco-Evasion Hypothesis. Glycobiology 22:1019–1030. doi: 10.1093/glycob/cws070 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laker R. C., Henry B. A., Wadley G. D., Clarke I. J., Canny B. J., and McConell G. K.. . 2011. Central infusion of leptin does not increase AMPK signaling in skeletal muscle of sheep. Am. J. Physiol. Regul. Integr. Comp. Physiol. 300:R511–R518. doi: 10.1152/ajpregu.00079.2010 [DOI] [PubMed] [Google Scholar]
  31. Landman R. E., Puder J. J., Xiao E., Freda P. U., Ferin M., and Wardlaw S. L.. . 2003. Endotoxin stimulates leptin in the human and nonhuman primate. J. Clin. Endocrinol. Metab. 88:1285–1291. doi: 10.1210/jc.2002-021393. [DOI] [PubMed] [Google Scholar]
  32. Lees P., and Taylor P. M.. . 1991. Pharmacodynamics and pharmacokinetics of flunixin in the cat. Br. Vet. J. 147:298–305. doi: 10.1016/0007-1935(91)90002-5 [DOI] [PubMed] [Google Scholar]
  33. Luheshi G. N., Gardner J. D., Rushforth D. A., Loudon A. S., and Rothwell N. J.. . 1999. Leptin actions on food intake and body temperature are mediated by IL-1. Proc. Natl. Acad. Sci. USA 96:7047–7052. doi: 10.1073/pnas.96.12.7047 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McKellar Q. A., Galbraith E. A., Bogan J. A., Russell C. S., Hooke R. E., and Lees P.. . 1989. Flunixin pharmacokinetics and serum thromboxane inhibition in the dog. Vet. Rec. 124:651–654. doi: 10.1136/vr.124.25.651 [DOI] [PubMed] [Google Scholar]
  35. Morrison C. D., Daniel J. A., Holmberg B. J., Djiane J., Raver N., Gertler A., and Keisler D. H.. . 2001. Central infusion of leptin into well-fed and undernourished ewe lambs: Effects on feed intake and serum concentrations of growth hormone and luteinizing hormone. J. Endocrinol. 168:317–324. doi: 10.1677/joe.0.1680317 [DOI] [PubMed] [Google Scholar]
  36. Murata H., Shimada N., and Yoshioka M.. . 2004. Current research on acute phase proteins in veterinary diagnosis: An overview. Vet. J. 168:28–40. doi: 10.1016/S1090-0233(03)00119-9. [DOI] [PubMed] [Google Scholar]
  37. National Research Council. 2007. Nutrient requirements of small ruminants: Sheep, goats, cervids, and new world camelids. China Legal Publ. House, Beijing, China. [Google Scholar]
  38. Petersen H. H., Nielsen J. P., and Heegaard P. M.. . 2004. Application of acute phase protein measurements in veterinary clinical chemistry. Vet. Res. 35:163–187. doi:10.1051/vetres:2004002. [DOI] [PubMed] [Google Scholar]
  39. Poüs C., Giroud J. P., Damais C., Raichvarg D., and Chauvelot-Moachon L.. . 1990. Effect of recombinant human interleukin-1 beta and tumor necrosis factor alpha on liver cytochrome P-450 and serum alpha-1-acid glycoprotein concentrations in the rat. Drug Metab. Dispos. 18:467–470. [PubMed] [Google Scholar]
  40. Ranjan R., Roy B. K., Ranjan A., and Singh S. K.. . 2011. Effect of E. coli endotoxin induced fever on the pharmacokinetic profile and dosage regimen of ceftriaxone in sheep (Ovis aries). Vet. Arh. 81:423–432. [Google Scholar]
  41. Sartin J. L., Marks D. L., McMahon C. D., Daniel J. A., Levasseur P., Wagner C. G., Whitlock B. K., and Steele B. P.. . 2008. Central role of the melanocortin-4 receptors in appetite regulation after endotoxin. J. Anim. Sci. 86:2557–2567. doi: 10.2527/jas.2008-0916 [DOI] [PubMed] [Google Scholar]
  42. Skibicka K. P., and Grill H. J.. . 2009. Hindbrain leptin stimulation induces anorexia and hyperthermia mediated by Hindbrain Melanocortin Receptors. Endocrinology 150:1705–1711. doi: 10.1210/en.2008-1316 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Soliman M., Abdelhady S., Fattouh I., Ishioka K., Kitamura H., Kimura K., and Saito M.. . 2001. No alteration in serum leptin levels during acute endotoxemia in sheep. J. Vet. Med. Sci. 63:1143–1145. doi: 10.1292/jvms.63.1143 [DOI] [PubMed] [Google Scholar]
  44. Soliman M., Ishioka K., Kimura K., Kushibiki S., and Saito M.. . 2002. Plasma leptin responses to lipopolysaccharide and tumor necrosis factor alpha in cows. Jpn. J. Vet. Res. 50:107–114. [PubMed] [Google Scholar]
  45. Sun Y., Yang Y., Qin Z., Cai J., Guo X., Tang Y., Wan J., Su D. F., and Liu X.. . 2016. The acute-phase protein orosomucoid regulates food intake and energy homeostasis via leptin receptor signaling pathway. Diabetes 65:1630–1641. doi: 10.2337/db15-1193 [DOI] [PubMed] [Google Scholar]
  46. Turek V. F., Olster D. H., Gililland K. R., Sheehy M., Ettenberg A., and Carlisle H. J.. . 2004. The effects of melanocortin agonists leptin-induced fever in and antagonists on rats. J. Therm. Biol. 29:423–430. doi: 10.1016/j.jtherbio.2004.08.011 [DOI] [Google Scholar]
  47. Whitlock B. K., Daniel J. A., Wilborn R. R., Maxwell H. S., Steele B. P., and Sartin J. L.. . 2010. Interaction of kisspeptin and the somatotropic axis. Neuroendocrinology 92:178–188. doi: 10.1159/000318049 [DOI] [PubMed] [Google Scholar]
  48. Zieba D. A., Amstalden M., and Williams G. L.. . 2005. Regulatory roles of leptin in reproduction and metabolism: A comparative review. Domest. Anim. Endocrinol. 29:166–185. doi: 10.1016/j.domaniend.2005.02.019 [DOI] [PubMed] [Google Scholar]

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