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Adipose Tissue Fat Accumulation Is Reduced by a Single Intraperitoneal Injection of Peroxisome Proliferator-Activated Receptor Gamma Agonist When Given to Newly Hatched Chicks

K. Sato^*,1, K. Matsushita, Y. Matsubara, T. Kamada^* and Y. Akiba

^* Department of Biological Production, Tokyo University of Agriculture and Technology, Japan 183-8509; Yamanashi Prefectural Livestock Experiment Station, Koufu, Japan 409-3812; and Graduate School of Agricultural Science, Tohoku University, Sendai, Japan 981-8555

¹ Corresponding author: satokan{at}cc.tuat.ac.jp

	ABSTRACT

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Peroxisome proliferator-activated receptor gamma (PPAR) is a transcription factor that regulates adipocyte differentiation and modulates lipid metabolism in mammals. The aim of the present study was to investigate whether the administration of PPAR ligands, adipogenic cocktail, or both to newly hatched chicks regulates adipocyte differentiation in vivo and modulates fat deposition in growing broiler chickens. Levels of PPAR, CCAAT/enhancer binding protein , and adipocyte fatty acid-binding protein mRNA in the abdominal fat pad of 7-d-old broiler chicks given a single intraperitoneal dose of troglitazone, a synthetic PPAR ligand, at 1 d old were significantly greater than those in control chickens. This suggests administration of troglitazone enhanced adipocyte differentiation in vivo. Adipose tissue weight in 28-d-old chickens similarly administered triolein emulsion containing troglitazone or adipogenic cocktail (i.e., dexamethasone, insulin, isobutyl-methylxanthine, and oleic acid) was also significantly less than that of control chickens. However, there was no significant difference in BW between treated and control chickens. Although BW and carcass composition were not different between troglitazone-treated and control chickens, at 48 d of age abdominal fat pad weight and feed intake were significantly decreased in chickens treated with troglitazone compared with controls. These results demonstrate that a single intraperitoneal injection of troglitazone to newly hatched chicks reduces fat deposition in mature broiler chickens.

Key Words: peroxisome proliferator-activated receptor • chicken • hyperplasia • fat deposition

	INTRODUCTION

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Obesity is a nutritional disorder prevalent in humans and other species, including chickens. Increased adipose tissue mass results from the multiplication of new fat cells through adipogenesis, from increased deposition of cytoplasmic triglycerides, or both (Soukas et al., 2001). In studying the development of adipose tissue in chickens, it has previously been shown that increases in the abdominal fat pad mass of broiler chickens mainly depend on hyperplasia of adipocytes until 4 wk of age and from then on hypertrophic growth (Hood, 1982). During hypertrophic growth, lipoprotein lipase (LPL)-catalyzed hydrolysis of triacylglycerols in the adipose tissue is the rate-limiting step in the accumulation of fat (Sato et al., 1999). Because in avians lipogenic activity is much greater in the liver than in adipose tissue (Griffin and Hermier, 1988; Sato et al., 1999), transport and incorporation of exogenous lipids (i.e., plasma very low-density lipoprotein and portomicrons) are essential for the deposition of cytoplasmic triglycerides in abdominal adipose tissue.

We previously reported that abdominal fat pad weights in 5-wk-old broiler chickens orally administered troglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPAR ) ligand, were significantly increased relative to those of control chickens, due to stimulation of PPAR activity and increases in LPL mRNA levels in abdominal adipose tissues (Sato et al., 2004). The PPAR plays a crucial role in hypertrophic growth of abdominal adipose tissue in chickens and is a key regulator in the early stages of chicken in vitro preadipocyte differentiation (Matsubara et al., 2005; Wang et al., 2008). However, the effects of administration of PPAR ligand on the hyperplasia stage of chicken adipose tissue deposition, starting from a few days before hatching until around 4 wk of age, have not been previously reported. To explore novel methods to control chicken fat deposition, the hyperplasia stage should also be targeted, and by controlling the activity of PPAR may prove to be an efficient mechanism for manipulating fat deposition in chickens.

In the present study, we investigated whether a single intraperitoneal injection of PPAR ligand, adipogenic cocktail, or both to newly hatched chicks modulates fat deposition in growing broiler chickens (i.e., when administered early in the adipocyte hyperplasia stage of fat deposition).

	MATERIALS AND METHODS

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Birds

Male broiler chickens (Ross strain, Matsumoto Hatchery, Zao, Japan) were housed in a controlled-temperature room and fed a commercial starter diet (d 0 to 21; CP 23%, ME 3,100 kcal/kg), and then a finisher diet (d 22 to 48; CP 19%, ME 3,200 kcal/kg). All procedures were approved by the Animal Care and Use Committee of the Graduate School of Agricultural Science of Tohoku University.

Quantitation of mRNA using Real-Time PCR

Total RNA was extracted from chicken abdominal adipose tissue using Trizol reagent (15596-018, Invitrogen, Carlsbad, CA). To study the expression of particular chicken adipogenesis genes, real-time reverse transcription-PCR analysis was performed using the iCycler Real Time Detection System (Bio-Rad Laboratories, Hercules, CA). The reverse transcription, amplification, and detection methods used were as described previously (Matsubara et al., 2005; Seol et al., 2006). At the end of each run, melting curve profiles were recorded. Analysis of the standard curve from each product allowed calculation of the mRNA levels of the respective genes. The sequences of the sense and anti-sense primers were as follows:

18S ribosomal RNA, estimated product size 312 bp (GenBank accession number AF173612 [GenBank] ):

5'-TAGATAACCTCGAGCCGATCGCA-3' and 5'-GACTTGCCCTCCAATGGATCCTC-3'.

PPAR', estimated product size 470 bp (GenBank accession number AB045597 [GenBank] ):

5'-TACATAAAGTCCTTCCCGCTGAC-3' and 5'-TC-CAGTGCGTTGAACTTCACAGC-3'.

CCAAT/enhancer binding protein (C/EBP), estimated product size 191 bp (GenBank accession number X66844 [GenBank] ):

5'-GTGCTTCATGGAGCAAGCCAA-3' and 5'-TGTC-GATGGAGTGCTCGTTCT-3'.

Adipocyte fatty acid-binding protein (aP2), estimated product size 107 bp (GenBank accession number AF432507 [GenBank] ):

5'-GAGTTTGATGAGACCACAGCAGA-3' and 5'-ATAACAGTCTCTTTGCCATCCCA-3'.

Results are presented as the ratio of each gene to 18S ribosomal RNA to correct for differences in the amounts of template DNA used.

Experiments 1, 2, 3, 4, and 5

Experiment 1. To investigate the effect of PPAR activation during the hyperplastic growth phase of adipocytes, 1-d-old male broiler chicks (Ross strain, Matsumoto Hatchery, Zao, Japan) were given a single intraperitoneal injection of the synthetic agonist troglitazone (Daiichi-Sankyo Pharmaceuticals, Tokyo, Japan; 1 mg/bird in PBS, volume 0.2 mL, n = 6). Control birds were injected with vehicle alone (volume 0.2 mL, n = 6).They were then kept in heated cages at 34°C for 1 d. All chicks were reared under conventional conditions and given commercial starter and finisher diets until 28 d of age. The housing temperature was gradually lowered to 24°C at 14 d of age. At 28 d of age, chickens were killed by cervical dislocation and abdominal fat pad weights measured.

Experiment 2. To assess the effect of vehicle on agonist efficacy in regulation of fat pad mass, triolein emulsion (Sato et al., 2002) was instead used for solubilization of the troglitazone. In brief, triolein (70 mg), cholesteryl oleate (1 mg), and phosphatidylcholine (10 mg) were taken to dryness with nitrogen and then suspended in 2 mL of 10 mM phosphate buffer containing 0.15 M NaCl, pH 7.4. The mixture was then subjected to sonification at 50 W (5 x 1 min with 30-s intervals for cooling) and the resultant particles isolated by KON-TRON centrifugation at 50,000 x g for 20 min at 4°C. Troglitazone (1 mg) was mixed with the triolein emulsion (1 mg of triacylglycerol) and incubated at 37°C for 30 min. One-day-old male chicks (Ross, Matsumoto Hatchery, Zao, Japan) were intraperitoneally injected with triolein emulsion containing troglitazone (1 mg/bird, volume 0.2 mL) or triolein emulsion alone (volume 0.2 mL; n = 5 in each group). All chicks then received commercial broiler starter and finisher diets until 28 d of age and were housed under similar temperature conditions as in experiment 1. At 28 d of age, chickens were killed by cervical dislocation and abdominal fat pad weights measured.

Experiment 3. To investigate the gene expression profiles resulting from a single injection of troglitazone to newly hatched chicks, age-dependent changes in adipose tissue gene expression were examined. One-day-old male chicks (Ross, Matsumoto Hatchery, Zao, Japan) were intraperitoneally administered triolein emulsion containing troglitazone (1 mg/bird, volume 0.2 mL) or triolein emulsion alone (volume 0.2 mL; n = 8 in each group), and were kept in heated cages at 34°C for 1 d. The temperature was gradually lowered to 24°C at 14 d of age. Diet and water were freely provided and chickens housed in similar conditions as in experiment 1. At 7 and 14 d of age, 4 chickens from each group were killed by cervical dislocation and abdominal fat samples collected and frozen in liquid nitrogen. The tissues were stored at –80°C until analysis. The levels of PPAR, C/EBP, and aP2 mRNA, which are marker of adipocyte differentiation, were then analyzed by real-time reverse transcription-PCR as described above.

Experiment 4. To confirm that enhancement of adipocyte differentiation in newly hatched chicks effectively manipulates fatness in broiler chickens at 28 d of age, 1-d-old male broiler chicks (Ross, Matsumoto Hatchery, Zao, Japan) were intraperitoneally given either triolein emulsion containing adipogenic cocktail (the mixture of 4 µg of dexamethasone, 2 µg of insulin, 4 µg of isobutyl-methylxanthine, and 50 µg of oleic acid to each bird, volume 0.2 mL) or triolein emulsion alone (volume 0.2 mL; n = 4 in each group). All chicks then received commercial broiler starter and finisher diets until 28 d of age and were housed under the same conditions as in experiment 2. At 28 d of age, chickens were killed by cervical dislocation and abdominal fat pad weights measured.

Experiment 5. To investigate the effect on growth performance and carcass composition of broiler chickens of a single injection of troglitazone to newly hatched chicks, 140 one-day-old male broiler chicks (Ross, Matsumoto Hatchery, Zao, Japan) were randomly assigned to floor pens and intraperitoneally administered triolein emulsion containing troglitazone (1 mg/bird, volume 0.2 mL) or triolein emulsion alone (volume 0.2 mL; n = 75 in each group). At 48 d of age, birds were killed, and growth performance, carcass composition, and abdominal fat pad weights were measured.

Statistical Analyses

The SAS applications package was used for statistical calculations (SAS version 6.03, SAS Institute Inc., Cary, NC). Results are shown as means ± SD. Statistical significance was determined using the Student’s t-test with 2-tailed P-values. The level of significance used in all studies was P < 0.05.

	RESULTS

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Effects on Fat Deposition of a Single Troglitazone Injection During the Hyperplasia Stage of Fat Accumulation as Observed at 28 d of Age (Experiments 1 and 2)

The abdominal fat pad weight of chickens given a single intraperitoneal injection of PBS containing 1 mg of troglitazone (n = 6) when 1 d old, measured at 28 d of age, was not significantly decreased vs. control chickens (P = 0.08). Their BW did not significantly differ (1,456 ± 31 vs. 1,453 ± 39 control vs. treated chickens; Table 1). The solubilization of troglitazone using triolein emulsion enhanced the drugs’ reduction in chickens. The adipose tissue weight of 28-d-old chickens administered triolein emulsion containing troglitazone when 1 d old was significantly less than that of control chickens administered triolein emulsion only, although BW was not significantly different between control and treated chickens (1,513 ± 81 and 1,475 ± 74, respectively; Table 1).

Levels of PPAR mRNA in the abdominal adipose tissue of chickens administered troglitazone (treated) were found to be significantly greater than those of control chickens at 7 d of age (Figure 1). However, levels at 14 d of age were not significantly different between control and treatment chickens.

View larger version (14K): [in this window] [in a new window]   Figure 1. Levels of peroxisome proliferator-activated receptor (PPAR; A), CCAAT/enhancer binding protein (C/EBP; B), or adipocyte fatty acid-binding protein (aP2; C) mRNA in adipose tissue of broiler chickens at 7 or 14 d of age (experiment 3). One-day-old male broiler chicks were injected intraperitoneally with 0.2 mL of either triolein emulsion containing 1 mg of troglitazone (treated) or triolein emulsion only (control), and the abdominal fat pad of 4 chickens per group was collected at 7 or 14 d of age. The expression of each gene was determined by real-time reverse transcription-PCR and was expressed as a ratio to 18S levels. Bars indicate SD of the mean (n = 4). The statistical superscripts are only for comparisons within a time period (e.g., 7 or 14 d) within a gene. a,bBars with different letters are significantly different (P < 0.05).

Effect of a Single Injection of Adipogenic Cocktail During the Hyperplasia Stage on Fat Deposition at 28 d of Age (Experiment 4)

The 28-d-old abdominal adipose tissue weight of chickens administered triolein emulsion containing adipogenic cocktail specific for chicken adipocytes when 1 d old was significantly less than that of control chickens administered triolein emulsion alone, although BW did not significantly differ between control and treated chickens (1,382 ± 43 and 1,334 ± 105 g, respectively; Table 2).

Body weight and carcass composition at 21 and 48 d of age were not significantly different between control and chickens administered triolein emulsion containing troglitazone at 1 d of age. However, abdominal fat pad weight and feed intake were significantly decreased in chickens treated with the PPAR agonist (Tables 3 and 4). Thus, the feed conversion rate was markedly improved by a single intraperitoneal administration of troglitazone to 1-d-old chicks (Table 4).

	DISCUSSION

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We previously reported that culture of chicken pre-adipocytes in differentiation medium supplemented with adipogenic cocktail (i.e., dexamethasone, insulin, and isobutyl-methylxanthine) and oleate resulted in their rapidly increased PPAR expression, greatly increased intracellular lipid deposition, increased aP2 (an adipocyte fatty acid binding protein), mRNA levels, and increased glycerol-3-phosphate dehydrogenase activity (Matsubara et al., 2005). This result showed that supplementation of adipogenic cocktail to the culture media of chicken pre-adipocytes strongly induced adipocyte differentiation. We investigated in the present study whether a single injection of adipogenic cocktail could provide direct evidence of the relationship between adipocyte differentiation and fat deposition during adipose tissue development in vivo (Wu et al., 2000; Matsubara et al., 2005) (experiment 4). Our current results clearly demonstrated that the abdominal fat pad weights of 28-d-old chickens were significantly decreased by a single intraperitoneal injection of adipogenic cocktail to 1-d-old chicks compared with control chickens (Table 2). In addition, the levels of aP2 mRNA in the abdominal fat pad of 7-d-old broiler chicks given a single intraperitoneal dose of adipogenic cocktail at 1 d old were significantly greater than those in control chickens as similar to troglitazone injection [0.00013 ± 0.00005 vs. 0.00531 ± 0.00124 (aP2/18S) in control vs. treated chickens; data not shown]. Therefore, the present results provide direct evidence of decreased fat deposition in chickens administered the troglitazone, a PPAR ligand, and results in the enhancement of differentiation in the hyperplasia growth stage of chicken adipocytes. This possible effect may be substantiated by the changes in levels of PPAR and C/EBP mRNA in the abdominal adipose tissue of broiler chickens administered troglitazone. The PPAR and C/EBP appear to cross-regulate each other (Schwarz et al., 1997), and upregulation of PPAR and C/EBP gene expression is known to induce adipogenesis in 3T3-L1 cells (Mandrup and Lane, 1997). In our previous in vitro study of chicken adipocytes, rapid increases in PPAR expression were observed after 9 h of culture in differentiation medium, and C/EBP mRNA reached maximum levels in chicken adipocytes after 24 h of culture in the differentiation media (Matsubara et al., 2005). Our present study shows that PPAR and C/EBP mRNA levels in adipose tissue of chickens administered troglitazone were significantly greater than those of control chickens at 7 d of age (Figure 1). Taken together, it can be inferred that the administration of PPAR ligand to 1-d-old chicks enhances adipocyte differentiation in vivo, resulting in decreased abdominal fat deposition after 28 d.

As troglitazone is poorly water-soluble (Dressman and Reppas, 2000), it is essential to solubilize the troglitazone well to enhance the absorption and reaction of the drug after administration. An amphipathic carrier (e.g., micelle, emulsion, liposome) is usually required for the dispersal of poorly water-soluble drugs when in aqueous solution for administration (Perkins et al., 2000). Residual yolk sac lipoprotein, which contains triacylglycerol, cholesterol ester, and phospholipids, is an important nutrient source for newly hatched chicks (Murakami et al., 1992; Moran, 2007). Thus, we used triolein emulsion (Sato et al., 2002), which has a similar profile of lipoproteins, for solubilization of the troglitzone. The solubilization of troglitazone using triolein emulsion significantly reduced the fat deposition of chickens (Table 1). In contrast, the abdominal fat pad weight of chickens given a single intraperitoneal injection of PBS containing 1 mg of troglitazone when 1 d old, measured at 28 d of age, was not significantly decreased versus control chickens (Table 1), and the result was confirmed through a similar experiment conducted successively, but the data were not present in this paper. These results, therefore, suggest that the preparation of emulsion for poorly water-soluble drugs, such as troglitazone, may augment the effect of these drugs upon cells.

Gene polymorphism of aP2 is associated with abdominal fat weight and percentage of abdominal fat. The aP2 gene could be a candidate locus or may be linked to a major gene that affects abdominal fat content in chickens (Wang et al., 2007). In the present study, aP2 mRNA expression in abdominal adipose tissue of broiler chickens administered troglitazone was found to be significantly greater than that of control chickens at both 7 and 14 d of age (Figure 1). This result suggests that aP2 is also involved in controlling fat deposition in broiler chickens. In conclusion, a single intraperitoneal injection of troglitazone to newly hatched chicks induces adipocyte differentiation in vivo and thus reduces fat deposition in broiler chickens.

	ACKNOWLEDGMENTS

 
We thank M. Suzuki of Sankyo Pharmaceuticals, Tokyo, Japan, for providing the troglitazone. This work was partly supported by grants-in-aid (No. 18380163 and 18580278) from the Ministry of Education, Science and Culture of Japan.

Received for publication February 25, 2008. Accepted for publication July 14, 2008.

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