Comparative Biochemistry and Physiology, Part A 176 (2014) 59–64
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Cholesterol deregulation induced by chronic corticosterone (CORT) stress in pectoralis major of broiler chickens Yujing Duan, Wenyan Fu, Song Wang, Yingdong Ni ⁎, Ruqian Zhao Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China
a r t i c l e
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Article history: Received 1 April 2014 Received in revised form 18 June 2014 Accepted 9 July 2014 Available online 16 July 2014 Keywords: Corticosterone Cholesterol metabolism Pectoralis major muscle Broilers
a b s t r a c t Chronic endogenous glucocorticoid (GC) excess in mammals is associated with metabolic dysfunction and dyslipidemia that are characterized by increased plasma triglyceride and total cholesterol (Tch) levels. However, the effects of chronic GC administration on cholesterol metabolism, particularly in muscle tissues of broiler chickens, are unknown. In this study, broiler chickens were treated chronically with vehicle (CON) or corticosterone (CORT) for 2 weeks. Chronic CORT treatment signiﬁcantly increased Tch levels in pectoralis major muscle (PMC) (p b 0.001) as well as in leg muscle (p b 0.01), and CORT enhanced triglyceride levels in the PMC (p b 0.001). Real-time PCR results showed that HMGCR (p b 0.05) mRNA expression was up-regulated by CORT in PMC, and 11β-HSD1 gene transcription (p = 0.08) was not signiﬁcantly downregulated, whereas glucocorticoid receptor (GR) mRNA expression, 11β-HSD2, CYP7A1, CYP27A1, ApoB and LDLR were unchanged by CORT (p N 0.05). Western blot results showed that the levels of total GR (p = 0.08) tended to be increased and nuclear GR protein (p b 0.05) was increased in PMC by CORT administration. Parallel to an increase in gene expression, HMGCR protein expression in PMC was signiﬁcantly increased (p b 0.05) by CORT. Moreover, LDLR (p b 0.05), ApoA1 (p = 0.06) and 11β-HSD2 (p = 0.07) protein expression in PMC tended to be increased by CORT compared to control. These results indicate that chronic CORT administration causes cholesterol accumulation in PMC tissues of broiler chickens by increasing cholesterol synthesis and uptake. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Hyperlipidemia and metabolic abnormalities caused by endogenous glucocorticoid (GC) excess have been well documented in mammals and birds. Much information is available regarding long-term dexamethasone (DEX) induction of common metabolic dysfunction and dyslipidemia characterized by increased fasting plasma triglyceride and total and low-density lipoprotein cholesterol (LDLC) concentrations, and decreased high-density lipoprotein cholesterol (HDLC) concentration. In mammals, increased circulating GCs together with the altered insulin sensitivity are suggested to be responsible for enhanced visceral fat deposition and hyperlipidemia (Geraert et al., 1996). In
Abbreviations: 11β-HSD, 11β-hydroxysteroid dehydrogenase; APOA1, apoliprotein A1; ApoB, apoliprotein B; CORT, corticosterone; CYP7A1, cholesterol-7-alpha hydroxylase; CYP27A1, sterol 27-hydroxylase; DEX, dexamethasone; GC, glucocorticoid; GR, glucocorticoid receptor; HDLC, high-density lipoprotein cholesterol; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; LDLC, low-density lipoprotein cholesterol; LDLR, lowdensity lipoprotein receptor; LDLs, low-density lipoproteins; PMC, pectoralis major muscle; Tch, total cholesterol; TG, total triglycerides. ⁎ Corresponding author at: Key Laboratory of Animal Physiology and Biochemistry, Nanjing Agricultural University, Nanjing 210095, China. Tel.: + 86 2584399020; fax: +86 2584398669. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.cbpa.2014.07.010 1095-6433/© 2014 Elsevier Inc. All rights reserved.
previous studies, GC administration increased hepatic lipogenesis and triglyceride accumulation in adipose tissues in rats (Bowes et al., 1996). In chicken, exogenous GC administration increased hepatic lipogenesis as well as intramyocellular lipid uptake and accumulation in broiler chickens (Lin et al., 2004; Wang et al., 2010; Wang et al., 2012). Our previous study demonstrated that chronic corticosterone (CORT) administration did not change plasma lipid proﬁle, except for a moderate increase of HDLC levels in broiler chickens (Wang et al., 2013). However, until now, data regarding cholesterol metabolic status in skeletal muscle has not been available. Cholesterol levels in tissues reﬂect a balance among dietary uptake, endogenous de novo synthesis, efﬂux, and utilization to bile acids (Faust and Kovacs, 2014). A previous study focused on HMGCR transcription level in the liver, breast or thigh muscles in Beijing-you chickens whose 3′-untranslated region (UTR) of the HMGCR gene was mutated (Cui et al., 2010). With respect to cholesterol uptake, the LDL receptor (LDLR) and ApoA1 are well-known for their important roles in regulating plasma and intracellular cholesterol homeostasis (Soto-Acosta et al., 2013); they are primarily modulated by intracellular cholesterol levels (Liu et al., 2012). Two key factors, cholesterol-7-alpha hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1), are prominently involved in the biosynthesis of bile acid from cholesterol and participate in the degradation of cholesterol in the liver (Bjorkhem et al., 2002). However, information regarding HMGCR,
Y. Duan et al. / Comparative Biochemistry and Physiology, Part A 176 (2014) 59–64
LDLR and sterol hydroxylase in the regulation of cholesterol metabolism in skeletal muscle following CORT-treatment is scarce. CORT conveys its signals primarily through the glucocorticoid receptor (GR), which is the target of endogenous CORT and certain synthetic steroids (Goodwin et al., 2013). As a nuclear hormone receptor, GR is widely conserved and presented in most organs; it is involved in both healthy and disease conditions (Stolte et al., 2006). The intracellular levels of active GC are regulated by several GC-metabolizing enzymes. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates, whereas 11β-HSD2 inactivates GCs (Holmes and Seckl, 2006; Tomlinson et al., 2004). In commercial production, chickens are subjected to a number of stressors prior to slaughter, including feed deprivation, crating density and transportation, which results in a negative impact on meat quality (Delezie et al., 2007). The serum concentrations of total and LDLcholesterol were increased but serum HDL-cholesterol decreased in heat stress broilers (Habibian et al., 2013). As an essential component of cell structure and the precursor of steroid hormone, the amount of cholesterol in chicken muscle will affect avian well-being, and may ultimately inﬂuence human health through dietary intake. It has been reported that, in humans, dietary intake of cholesterol and saturated fatty acids is strongly associated with coronary heart disease and arteriosclerosis (Simopoulos, 2006). Moreover, the muscle plays a critical role in maintaining systemic energy homeostasis and accounts for about 80% of insulin-directed glucose disposal (Nguyen et al., 2014). Investigating the effect of GC on cholesterol metabolism in skeletal muscle in chickens would help elucidate the mechanism of intramyocellular cholesterol accumulation. Therefore, the objective of the present study was to investigate the effect of chronic CORT administration via drinking water on cholesterol metabolism in pectoralis major muscle (PMC), and to clarify the underlying mechanism through the measurement of gene and protein expressions involved in the metabolic process. 2. Materials and methods 2.1. Ethics statement All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Agricultural University and the sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China and “the Regulation regarding the Management and Treatment of Experimental Animals” (2008) No. 45 set by the Jiangsu Provincial People's Government.
NanoDrop ND-1000 Spectrophotometer (Thermo, USA). The ratios of absorption (260/280 nm) of all samples were between 1.8 and 2.0. Aliquots of RNA samples were subjected to electrophoresis through a 1.4% agarose–formaldehyde gel to verify their integrity. Two micrograms of total RNA was treated with RNase-Free DNase (M6101, Promega, USA) and reverse-transcribed according to the manufacturer's instructions. 2 μL of diluted cDNA (1:40, vol/vol) was used for real-time PCR which was detected in Mx3000P (Stratagene, USA). β-Actin, which is not affected by the experimental factors (CORT) (Duan et al., 2013), was chosen as the reference gene. All the primers chosen to study the expression of genes related to CORT and cholesterol metabolism, as listed in Table 1, were synthesized by Generay (Shanghai, China). The method of 2−ΔΔCt was used to analyze the real-time PCR results and gene mRNA levels were expressed as the fold change relative to the mean value of control group (Livak and Schmittgen, 2001).
2.4. Nuclear and total protein extracts and western blotting Nuclear protein extracts were prepared from the muscle as previously described (Rudiger et al., 2002; Sun et al., 2013). Total protein extracts were prepared as previously described (Duan et al., 2013). Protein concentrations were determined using a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Forty micrograms of protein extract from each sample was then loaded onto 7.5% SDS-PAGE gels and the separated proteins were transferred onto the nitrocellulose membranes (BioTrace, Pall Co, USA). After transfer, membranes were blocked for 2 h at room temperature in blocking buffer and then membranes were incubated with the following primary antibodies: GR (1:200; sc1004, Santa Cruz), 11β-HSD1 (1:200; sc20175, Santa Cruz), 11β-HSD2 (1:200; sc20176, Santa Cruz), HMGCR (1:200; sc33827, Santa Cruz), CYP7A1 (1:1000; ab65596, Abcam), CYP27A1 (1:500; BS2192, Bioworld, USA), APOA1 (1:500; BS06158, Bioworld, USA), LDLR (1:500; 10785-1-AP, Proteintech, USA), Lamin A/C (1:500; BS1446, Bioworld, USA), and GAPDH (1:10000; KC-5G4, Changchen, China), in dilution buffer overnight at 4 °C. After several washes in Tris-BufferedSaline with Tween (TBST), membranes were incubated with goat antirabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Bioworld, USA) in dilution buffer for 2 h at room temperature. After several washes, bands were visualized by enhanced chemiluminescence (ECL) using the LumiGlo substrate (Super Signal West Pico Trial Kit, Pierce, USA), and the signals were recorded by an imaging System (Bio-Rad, USA), and analyzed with Quantity One software (Bio-Rad, USA).
2.2. Animals and experimental design Experimental design and animal management have been well documented in the previous publication (Wang et al., 2013). In brief, broiler breeder eggs (Ross 308) were incubated under standard conditions. Newly hatched chickens received a starter diet (12.5 ME/kg; 21% CP) from 1 d to 20 d and a ﬁnish diet (12.8 ME/kg; 19.5% CP) from 21 d to 42 d and water was provided ad libitum throughout the experiment. The light was continuous during the ﬁrst week and decreased gradually to 18L:6D. On d27, the chickens were divided into control and CORTtreated groups. Chickens in CORT group were supplied with water supplemented with 5 mg/L CORT (C2505, SIGMA, USA), in contrast, those in control groups were supplied with water supplemented with equivalent volume of the solvent (absolute ethanol) for the following 15 d. On d42, all chickens were sacriﬁced by decapitation, and PMC and leg muscle samples were rapidly frozen in liquid nitrogen and then stored at −70 °C. 2.3. RNA isolation, cDNA synthesis and real-time PCR Total RNA was extracted from PMC samples with TRIzol Reagent (15596026, Invitrogen). Quantity of the RNA was measured by
2.5. Cholesterol and triglyceride contents in the muscle A modiﬁed procedure was used for extraction of total lipids from tissue samples, as described (Cong et al., 2012). Brieﬂy, 50 mg of frozen muscle sample was homogenized in 1 mL ice-cold buffer RIPA, then 200 μL of homogenates was homogenized with 800 μL mixture of chloroform/methanol (2:1, vol/vol) and centrifuged at 3000 g for 10 min. The bottom (chloroform) layer was removed, air-dried and reconstituted in 30 μL mixture of tert-butyl alcohol and methanol (13:2, vol/vol). The Tch content was determined by cholesterol assay kit (006301, Beijingbeihua, China), the triglyceride content was determined by triglyceride assay kit (006304, Beijingbeihua, China).
2.6. Statistical analysis The results are presented as means ± SEM. The general linear model was conducted to evaluate the effects of CORT. All analyses were performed using SPSS 18.0 software. p values less than 0.05 were considered statistically signiﬁcant.
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Table 1 Primer sequences for real-time PCR ampliﬁcation. Target genes
PCR products (bp)
GR 11β-HSD1 11β-HSD2 HMGCR CYP7A1 CYP27A1 ApoB LDLR β-Actin
DQ227738.1 NM001001201.1 XM003643178.2 NM_204485.1 AB109636.1 XM422056.4 NM_001044633.1 NM_204452.1 L08165.1
203 171 90 137 106 185 87 86 300
F: 5′-CTTCCATCCGCCCTTCA-3′ R: 5′-TCGCATCTGTTTCACCCC-3′ F: 5′-CACCTTCCAGCCAGAGCA-3′ R: 5′-CCGAGGGTCAGGCATTTC-3′ F: 5′-ACTTTGTCCGAGACTTGTTCCT-3′ R: 5′-GCTTCTTGGCGTCCTTGTG-3′ F: 5′-TTGGATAGAGGGAAGAGGGAAG-3′ R: 5′-CCATAGCAGAACCCACCAGA-3′ F: 5′-CATTCTGTTGCCAGGTGATGTT-3′ R: 5′-GCTCTCTCTGTTTCCCGCTTT-3′ F: 5′-AGGACTTTCGTCTGGCTCT-3′ R: 5′-CTCCGCATCGGGTATTT-3′ F: 5′-GCATCTCTGCATCTCAGGAAAGA-3′ R: 5′-GCAGGCTACAAACTAACAGATCCA-3′ F: 5′-CCACCATTTGGCAGAGGAA-3′ R: 5′-ACCGCAGTCAGACCAGAAGAG-3′ F: 5′-TGCGTGACATCAAGGAGAAG-3′ R: 5′-TGCCAGGGTACATTGTGGTA-3′
GR, glucocorticoid receptor; 11β-HSD, 11β-hydroxysteroid dehydrogenase; HMGCR, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; CYP7A1, cholesterol-7-alpha hydroxylase; CYP27A1, sterol 27-hydroxylase; ApoB, apoliprotein B; LDLR, low-density lipoprotein receptor.
CYP7A1 and CYP27A1, were not signiﬁcantly altered by CORT administration (p N 0.05).
3. Results 3.1. Chronic CORT treatment increases cholesterol and triglyceride contents in muscle tissues As shown in Fig. 1, CORT administration markedly enhanced cholesterol levels in both the PMC (p b 0.001) and leg muscle (p b 0.01). CORT signiﬁcantly increased total triglyceride (TG) in CORT-treated chickens in the PMC muscle (p b 0.001) but not in leg muscle (p N 0.05). 3.2. Expression of genes involved in CORT metabolism or signaling pathway at the mRNA level As shown in Table 2, HMGCR mRNA expression was signiﬁcantly upregulated in the PMC by CORT (p b 0.05) compared with control, and 11β-HSD1 mRNA expression tended to decrease (p = 0.08) in CORT treated group compared with control. However, the mRNA levels of other genes, including GR, 11β-HSD2, CYP7A1, CYP27A1, ApoB and LDLR, were not markedly altered by CORT administration (p N 0.05) in PMC tissues. 3.3. Protein levels of genes involved in CORT metabolism or signaling pathway
4. Discussion One striking effect of GC treatment is the marked enhancement of lipid accumulation in the plasma and liver (Petrovic et al., 1993). However, the effect of chronic CORT on cholesterol metabolism in skeletal muscle in chicken has not been reported. In the present study, a significant accumulation of cholesterol and triglycerides was observed for the ﬁrst time in skeletal muscle of broiler chickens after chronic CORT administration, which will be helpful for estimating chickens' wellbeing and meat quality. 4.1. Effect of CORT on GR-mediated response The GR-mediated response to CORT administration is critical for the maintenance of homeostasis (Chinenov and Rogatsky, 2007). Watts et al. found that a reduction in hepatic and adipose tissue GR expression using antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents (Watts et al., 2005). The decrease of the number of GRs in patients with type IIa hyperlipidemia appears to be
As shown in Fig. 2, CORT administration showed a tendency to increase GR protein level in whole-cell lysates (p = 0.08) and signiﬁcantly increased nuclear GR protein level in the PMC (p b 0.05) compared with controls. As shown in Fig. 3, 11β-HSD2 protein level in PMC tissues was moderately increased by CORT (p = 0.07), while 11β-HSD1 protein content in the PMC was not signiﬁcantly different between CORT and control broilers (p N 0.05). As shown in Fig. 4, signiﬁcant increases in both the mRNA and protein levels of HMGCR in PMC tissues were also observed after CORT administration (p b 0.05). LDLR and ApoA1 are two major factors involved in processing cholesterol uptake into cells, LDLR protein levels were signiﬁcantly increased (p b 0.05), and ApoA1 levels tended to increase (p = 0.06) in the PMC after CORT treatment. However, the protein levels of two major enzymes controlling cholesterol utilization, Table 2 Effects of CORT on mRNA content in PMC of broiler chickens. Items
GR 11β-HSD1 11β-HSD2 HMGCR CYP7A1 CYP27A1 ApoB LDLR
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
± ± ± ± ± ± ± ±
CORTb 0.10 0.07 0.07 0.10 0.38 0.23 0.26 0.12
Values are means ± SEM. n = 12/group. a CON = control. b CORT = corticosterone.
1.08 0.80 0.86 1.34 1.06 0.89 1.19 1.05
± ± ± ± ± ± ± ±
p-Value 0.07 0.07 0.07 0.09 0.24 0.13 0.16 0.24
NS 0.08 NS b0.05 NS NS NS NS Fig. 1. Effects of CORT on triglyceride and total cholesterol levels in leg muscle (A) and the PMC (B) of broiler chickens. CON = control; CORT = corticosterone. Data are expressed as the means ± SEM (n = 12).
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limiting enzyme, HMGCR (Garevik et al., 2012; Ness and Chambers, 2000). HMGCR activity is increased in cultured hepatocytes after DEX treatment, leading to increased cholesterol synthesis (Lin and Snodgrass, 1982). GC also elevates HMGCR activity in HeLa cells (Cavenee and Melnykovych, 1979). The chicken HMGCR gene is present in many tissues, including the brain, liver, intestine and skeletal muscle (Burtea et al., 1991). In the present study, cholesterol accumulation and signiﬁcant upregulation of HMGCR at the mRNA and protein levels in the PMC of CORT-treated broilers were observed, suggesting an increase in intramuscular cholesterol de novo synthesis. 4.3. Effect of CORT on cholesterol degradation
Fig. 2. Effects of CORT on GR protein expression in PMC of broiler chickens. Quantiﬁcation of the densitometry of total GR (A) and nucleus GR (B). These results were averaged from at least 12 immunoblots. CON = control; CORT = corticosterone. Data are normalized to the optical densities of GAPDH and Lamin C, respectively, and are expressed as the means ± SEM (n = 12).
a compensatory cellular response, culminating in the activation of endogenous cholesterol synthesis (Petrichenko et al., 1991). In the present study, higher levels of GR protein, especially in nucleus, were observed in the PMC of CORT treated chickens compared with controls, indicating that GR signaling cascade was activated. Moreover, 11β-HSD2 protein levels were also moderately upregulated by CORT administration. 11β-HSD2 is a high-afﬁnity dehydrogenase inactivating GC, and is highly expressed in placenta and fetus to prevent premature maturation of fetal tissues and consequent developmental programming (Chapman et al., 2013). A higher level of 11β-HSD2 protein in the muscle might contribute to inactivate CORT in muscle tissues of CORT-treated chickens. With respect to the increase of GR protein in PMC of CORT-treated broilers, no signiﬁcant change of GR mRNA expression was observed between control and treated broilers, which indicates the post-transcriptional regulation such as mRNA transport, turnover and translation, as reported in chicken hypothalamus (Wang et al., 2014). 4.2. Effect of CORT on cholesterol synthesis Intracellular cholesterol content depends on three major factors: cholesterol uptake into cells, de novo cholesterol synthesis within cells, and efﬂux of cholesterol out of cells (Feeney et al., 2013). With respect to de novo cholesterol synthesis, multiple mechanisms for the feedback control of cholesterol biosynthesis converge at the rate-
Fig. 3. Effects of CORT on HSD protein expression in the PMC of broiler chickens. Quantiﬁcation of the densitometry of 11β-HSD1 (A) and 11β-HSD2 (B). These results were averaged from at least 12 immunoblots. CON = control; CORT = corticosterone. Data are normalized to the optical density of GAPDH and are expressed as means ± SEM (n = 12).
Regarding cholesterol degradation, the liver is the only site in which substantial amounts of cholesterol are removed from the body either by excretion into bile as free cholesterol or after conversion into bile acids, a process regulated by the microsomal enzyme CYP7A1 (Rudling et al., 2002). CYP27A1 also participates in the degradation of cholesterol in the liver (Bjorkhem et al., 2002). DEX induces hepatic CYP7A1 and 27A1 gene expression and bile acid synthesis in rats (Liu et al., 2008). It also increases hepatic bile acid biosynthesis and CYP27A1-mediated enzyme activity in HepG2 cells (Tang et al., 2008). However, no data concerning the effects of GC on CYP7A1 and 27A1 are available. In the present study, the expression of CYP7A1 and 27A1 at the mRNA and protein levels was detected in the chicken PMC. However, neither mRNA nor protein levels were changed by chronic CORT administration. These results indicate that the breakdown of intracellular cholesterol in skeletal muscle was not altered by CORT administration in chicken. The distribution of CYP7A1 and 27A1 in skeletal muscle of chicken still needs further investigation. 4.4. Effect of CORT on cholesterol uptake The homeostasis of intracellular cholesterol in peripheral cells is primarily maintained via its inﬂux and efﬂux. Petrichenko et al. reported that DEX causes a net cellular inﬂux of free cholesterol into cell and decreases its efﬂux mediated by HDL from vascular smooth muscle cells (Petrichenko et al., 1997). Stein et al. observed that DEX decreases HDL-mediated cholesterol efﬂux from skeletal muscle in mice (Stein et al., 2004). Ayaori et al. indicated that the attenuated cholesterol efﬂux in macrophages contributes to GC-associated cardiovascular risk (Ayaori et al., 2006). The liver is a key organ in the control of plasma cholesterol, which is largely determined by the rate of removal of LDLs from the circulation via hepatic LDLR (Myant et al., 1991). The number of hepatic LDLRs directly governs plasma LDLC concentrations (Rudling et al., 2002). GC stimulates a concentration- and time-dependent increase of LDLR biosynthesis in cultured ﬁbroblasts (Filipovic and Buddecke, 1985). Receptor-mediated cholesterol uptake is suggested to play a role in maintaining the intracellular free cholesterol pool. In the current study, we observed that muscular LDLR and ApoA1 protein levels in the PMC of CORT-treated chickens were signiﬁcantly increased compared with controls. It was previously reported that ApoA1 is essential for the selective uptake of HDLC esters (Plump et al., 1996). It is important to note that high GC levels enhance lipid accumulation into macrophages cultured in vitro by increased cholesterol ester synthesis and decreased cholesterol ester breakdown without altering cholesterol inﬂux or efﬂux (Cheng et al., 1995). These results suggest that increased uptake of cholesterol into myocellular contributes to the accumulation of cholesterol in the PMC of chickens under chronic CORT treatment. In this study, we cannot exclude that the alterations of cholesterol esterization contribute to the intracellular cholesterol accumulation in the PMC in chicken induced by CORT treatment. In conclusion, chronic CORT administration induces cholesterol and triglyceride accumulation in chicken muscle by upregulating their intracellular synthesis and uptake.
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Fig. 4. Effect of CORT on key proteins involved in cholesterol homeostasis in the PMC of broiler chickens. Quantiﬁcation of the densitometry of HMGCR (A), CYP7A1 and CYP27A1 (B), and APOA1 and LDLR (C). These results were averaged from at least 12 immunoblots. CON = control; CORT = corticosterone. Data are normalized to the optical density of GAPDH and are expressed as the means ± SEM (n = 12).
Acknowledgment This work was supported by the Special Fund for Agro-scientiﬁc Research in the Public Interest (201003011), the Fundamental Research Funds for the Central Universities (KYZ201212) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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