Effects of dietary corticosterone on the central adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in broiler chickens

Abstract

Glucocorticoids (GCs) induce the activation of the central adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway in birds. In this study, we aimed to investigate the effects of corticosterone (CORT) supplemented in diet on the central AMPK signaling pathway in broilers. The average daily gain was reduced by CORT treatment, and the average daily feed intake remained unchanged. Plasma glucose, triglyceride, total cholesterol, and CORT contents were increased by CORT administration. In addition, CORT treatment decreased the relative weights of heart, spleen, and bursa and increased the relative weights of liver and abdominal fat. The glycogen contents in the liver and breast muscle were higher in the chicks treated with CORT. CORT treatment upregulated the gene expression of mammalian target of rapamycin, glucocorticoid receptor, AMPKα2, neuropeptide Y(NPY), liver kinase B1 (LKB1), AMPKα1, and fatty acid synthase in the hypothalamus. Moreover, CORT treatment increased the protein levels of acetyl-coenzyme A carboxylase (ACC) phosphorylation and total AMPK and phosphorylated AMPK in the hypothalamus. Hence, CORT administration in the diet activated the LKB1-AMPK-NPY/ACC signaling pathway in the hypothalamus of broiler.

Introduction

As the end product of the hypothalamus–pituitary–adrenal (HPA) axis (Song et al., 2011a), glucocorticoids (GCs) participate in the control of whole-body homeostasis and in the response to stress by stimulating the release of energy stores by promoting glucose (GLU) mobilization and lipolysis. Corticosterone (CORT), the main GC in birds, is involved in the control of appetite in poultry (Yuan et al., 2008). During exposure to stressors, the activation of the HPA axis was believed to be involved in depressed performance (Post et al., 2003). A previous study has shown that increased CORT release leads to remarkably low body weight gain (BWG) and feed utilization in the poultry (Lin et al., 2004a). At the same time, CORT administration leads to strengthened mobilization of muscle tissue and liver heterogenesis, thereby increasing blood GLU, free amino acid, and free fatty acid (FFA) levels (Lin et al., 2004a, 2004b; Dong et al., 2007).

The central nervous system (CNS) is involved in the control of energy homeostasis, hunger, and satiety feelings (Morton et al., 2006). The CNS can detect signals from the periphery regarding metabolic status, as well as metabolic processes, and integrate this information in a coordinated manner, thereby exerting thermal and energy regulatory roles (Serge et al., 2005). The hypothalamus and the brainstem are the main CNS regions responsible for the regulation of energy homeostasis in mammals (Sam et al., 2012). In mammals and avian species, the hypothalamus can integrate peripheral signals reflecting energy balance (Liu et al., 2016a). The arcuate nucleus (ARC) of the hypothalamus is believed to play a crucial role in the regulation of feeding (Chaudhri et al., 2008). The ARC of hypothalamus contains two populations of neurons with opposing effects on feed intake (Chaudhri et al., 2008).

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is a ubiquitously expressed sensor of cellular energy status (Hardie et al., 2012) that provides crucial input on brain cell adenosine triphosphate (ATP) availability to neural pathways governing whole-body energy stability (Xue and Kahn, 2006). Generally, AMPK is crucial in the regulation of whole-body energy balance (Hardie, 2015), which can be activated by the allosteric regulation of AMP and then by threonine (Thr172) phosphorylation on the α subunit in response to the changes in cellular energy status (Kahn et al., 2005). As proposed by Xue and Kahn (2006), AMPK in the hypothalamus and peripheral tissues coordinately integrates inputs from multiple sources to maintain energy homeostasis. Once exposed to stresses, such as metabolic poisons (arsenite and oligomycin), oxidative stresses, hypoxia, low GLU, muscle contraction, or nutrient deprivation, AMPK is activated, thereby depleting cellular ATP, leading to a concomitant increase in AMP and ultimately increasing feed intake (Kola et al., 2006). A previous study has shown that GCs might play an important regulatory role in peripheral and central AMPK activities in mammals (Nakken et al., 2010). Thus, we hypothesized that dietary CORT supplementation could regulate the central AMPK signaling pathway in broiler chickens.

The administration of CORT was used to mimic stress responses in our previous studies (Lin et al., 2004a, 2004b; Yuan et al., 2008; Liu et al., 2012). Lin et al. (2004a, 2004b) aimed to explore the effects of CORT on the induction of oxidative injury in broiler chickens. Meanwhile, Yuan et al. (2008) investigated the effects of CORT and dietary energy levels on feed or energy intake and fat deposition in broiler chickens. Although Liu et al. (2012) studied the effect of CORT on appetite-related genes, their study focused on laying hens. To the best of our knowledge, no study has determined the effect of dietary CORT on the central AMPK signaling pathway. This study aimed to characterize the effects of dietary CORT on the performance, plasma composition, and central AMPK signaling pathway in broiler chickens.

Materials and Methods

Broilers and housing

One-day-old male broiler chicks (Arbor Acres strain) were obtained from a local hatchery and reared in an environmentally controlled room. The brooding temperature was maintained at 35 °C at the beginning and then gradually decreased by 2 to 3 °C per week according to the age of broilers (Song et al., 2011b). The ambient humidity was 40% to 50 %. The light regime was 23:1 (L:D) h. The composition and the nutrient levels of the diet were shown in Supplementary Table S1. All of the birds were given free access to feed and water during the rearing period. This study was approved by the Shandong Agricultural University and conducted in accordance with the Guidelines for Experimental Animals of the Ministry of Science and Technology (Beijing, China).

Experimental design and sample collection

At 7 d old, 96 broiler chicks of similar body weights (BW) were randomly divided into two treatment groups with six replicates per treatment and eight chicks per replicate. The following treatments were randomly and separately applied to each group: 1) fed a basal diet and 2) CORT administration (30 mg/kg diet). The experiment lasted 7 d. Feed intake was recorded daily, and average daily gain (ADG) and average daily feed intake (ADFI) were measured at the end of the experimental period. At 14 d old, two chickens from each replicate were randomly selected for sample collection. Blood samples were collected using heparinized anti-coagulant tubes from the wing vein, then shaken gently, and transferred on ice. Then plasma was obtained after centrifugation at 400 × g for 10 min at 4 °C and stored at −20 °C for further analysis (Zhao et al., 2012; Huang et al., 2015). After obtaining blood samples, the chickens were slaughtered through the intravenous injection of pentobarbital sodium (30 mg/kg BW) and jugular exsanguination. The heart, liver, spleen, abdominal fat, and bursa were collected and weighed. Tissue samples were obtained from the hypothalamus, liver, breast muscle, and leg muscle. After washing with ice-cold sterilized saline solution, these samples were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis. The method for hypothalamus tissue collection followed reported procedures (Kuo et al., 2005; Tu et al., 2016; Hu et al., 2019) with some modification. After rapid decapitation, the hypothalamus was dissected from the ventral surface of the brain. The optic chiasm was carefully peeled off. Two transverse cuts were made at the apex of the optic chiasm and the rostral margin of the mammillary bodies. Bilateral cuts were then made 4 mm either side of the midline. The hypothalamus was dissected out (in sections 1 mm thick) along the bank of the third ventricle and preserved at −80 °C until use. One sample from each replicate plus two samples from the remaining six samples were used for analysis.

Measurements of plasma metabolites and glycogen content in muscle and liver

The concentrations of GLU (No. F006), urea (No. C013-2), total cholesterol (TCHO, No. A111), and triglycerides (TGs, No. A110) were measured spectrophotometrically via colorimetric enzymatic methods using commercial diagnostic kits (Jiancheng Bioengineering Institute, Nanjing, China). The plasma CORT concentration (ADI-900-097; Enzo Life Sciences, Farmingdale, NY, USA) was determined using a commercially available enzyme immunoassay kit according to the manufacturer’s instructions. The glycogen contents in liver, breast muscle, and leg muscle were measured by using commercial kits in accordance with the anthracenone method (A043; Jiancheng Bioengineering Institute).

RNA extraction and analysis

Gene expression in the hypothalamus was assessed using quantitative real-time polymerase chain reaction (PCR) with synergy brands (SYBR) Green I labeling as described previously (Liu et al., 2014; Tang et al., 2019). Total RNA from the hypothalamus was extracted using TRIzol Reagent (15596018; Invitrogen, San Diego, CA, USA). Extracted RNA was dissolved in RNase-free water and quantified by using a DeNovix spectrophotometer (DS-11; DeNovix Inc., Wilmington, DE, USA). RNA purity was verified by determining the absorbance ratio of 260 to 280 nm (OD260/280 = 1.8–2.0). RNA integrity was assessed through agarose gel electrophoresis.

Reverse transcription to complementary DNA (cDNA) was performed using a commercially available cDNA synthesis kit (RR037A; Takara Biotechnology, Dalian, China). reverse transcription (RT) reactions (20 µL) consisted of 1,000 ng of total RNA, 2 µL of 5 × gDNA Eraser Buffer, 1 µL of gDNA Eraser, 1 µL of PrimeScript RT Enzyme Mix I, 1 µL of RT Primer Mix, and 4 µL of 5 × PrimeScript Buffer 2 (for Real Time) and RNase Free dH₂O. All the chemicals were provided by the cDNA synthesis kit. Real-time analysis was conducted using a 7500 Real-time PCR System (Applied Biosystems, Foster, CA, USA) under the following protocol: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s. Primer set sequences are described in Supplementary Table S2. SYBR green fluorescence was detected at the end of each cycle to monitor the amount of PCR product. A standard curve was constructed using a 5-fold dilution series, and the slope was used to calculate the efficiency of the qPCR primers. The relative amount of messenger RNA (mRNA) for a gene was calculated in accordance with the method of Livak and Schmittgen (2001). The mRNA levels of target genes were normalized to β-actin (Δcycle threshold [CT]). ΔCT was calibrated against the average of the control chicks. The number of target molecules relative to the control was calculated by using 2^−ΔΔCT. Therefore, all gene transcription results are reported as the n-fold difference relative to the control. The results were verified using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. The specificity of the amplification product was verified by electrophoresis on 0.8% agarose gel and DNA sequencing.

Protein preparation and Western blotting

The determination of protein level was quantified according to the procedure described in Liu et al. (2014, 2018). The frozen hypothalami were treated with 1 mL of lysis buffer (P0013B; Beyotime Biotechnology, Shanghai, China) containing 10 µL of protease inhibitor and 10 µL of phosphatase inhibitor (FD1001, FD1002; Hangzhou Fude Biological Technology Co., Hangzhou, China) and homogenized by using an electrical homogenizer (Tissuelyser-24; Jingxin Industrial Development Co., Shanghai, China). Tissue homogenates were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatants were collected into fresh tubes. The protein concentration was assayed using a bicinchoninic acid protein assay kit (P0010; Beyotime Biotechnology). Each homogenate was mixed with 5 × SDS loading buffer and boiled at 100 °C for 10 min in a dry heater (100 °C). Equal amounts of total protein (80 µg) were electrophoresed in 7.5% to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Richmond, 246, CA). The separated proteins were then transferred electrophoretically to a polyvinylidene fluoride membrane (Millipore, USA) in Tris-glycine buffer (P0021B; Beyotime Biotechnology) containing 20% anhydrous ethanol using 200 mA at 4 °C for 2 h. After transfer, membranes were blocked in a commercial blocking buffer (P0023B; Beyotime Biotechnology) for 1 h at room temperature. The primary antibody was diluted in primary antibody dilution buffer (P0023A; Beyotime Biotechnology; 1:1,000). The membranes were incubated with primary antibodies at 4 °C with gentle shaking overnight. The primary antibodies used were phospho-mTOR^ser2448 (#2971), mTOR (#2983), phospho-AMPKα ^Thr172 (#2531), AMPKα (#2532), phospho-acetyl-coenzyme A carboxylase (ACC)^Ser79 (#3661) and ACC (#3662) (Cell Signaling Technology, Beverly, MA, USA), and anti-GAPDH (AG019; Beyotime Biotechnology). These antibodies were validated previously for use on chicken samples (Liu et al., 2014, 2016b). The blots were washed and incubated with horseradish peroxidase-linked anti-rabbit (A0208; Beyotime Biotechnology), anti-mouse (A0216; Beyotime Biotechnology) secondary antibodies for 4 h at 4 °C. After washing 10 min for three times with Tris-buffered saline plus 0.2% Tween-20, membranes were then visualized using enhanced chemiluminescence detection reagent (P0018; Beyotime Biotechnology). The membranes were then scanned using Fusion Fx (Vilber Lourmat SAS., Collégien, France), and signal intensities of the target proteins were calibrated by densitometry using Fusion-Capt Advance FX7 software optical density analysis (Vilber Lourmat SAS). The band intensity was normalized to the GAPDH band in the same sample.

Statistical analysis

The data were expressed as mean ± SEM. For growth performance analysis, n = 6 (six replicates). For other parameters, n = 8 (six from each replicate and two randomly selected from the remaining six samples). The two groups were compared via t-test. P < 0.05 was considered significant. SPSS version 11.0 software (SPSS, Chicago, IL, USA) was used for all statistical analyses.

Results

Performance and plasma parameters

The effects of CORT on broiler performance and plasma parameters are shown in Table 1. There was no difference in initial BW of 7-d-old broilers. Compared with the control condition, CORT treatment reduced the ADG (P < 0.05), but it had no effect on ADFI during the experiment (P > 0.1). In addition, CORT treatment increased the feed-to-gain ratio (F:G) compared with the control (P < 0.05). The concentrations of plasma GLU, TG, TCHO, and CORT were increased by CORT administration in 14-d-old chicks (P < 0.05).

Table 1.

Effects of CORT treatment on growth performance, plasma metabolites, organ relative weights, and glycogen contents of muscle and liver in chicks

	Treatment1
Items	Control	CORT	P-value
Growth performance2
Initial BW, g/bird	144.11 ± 2.10	144.65 ± 1.46	NS
Final BW, g/bird	380.21 ± 5.31a	280.10 ± 4.25b	<0.0005
ADG, g/d	33.73 ± 0.59a	19.35 ± 0.50b	<0.0005
ADFI, g/d	38.42 ± 0.65	38.42 ± 0.57	NS
F:G, g/g	1.14 ± 0.002b	1.99 ± 0.02a	<0.0005
Plasma metabolites3
GLU, mmol/L	11.66 ± 0.39b	13.86 ± 0.45a	0.0026
TG, mmol/L	0.49 ± 0.08b	2.26 ± 0.17a	<0.0005
TCHO, mmol/L	3.35 ± 0.16b	7.88 ± 0.87a	0.0002
CORT, pg/mL	489.44 ± 98.96b	4,582.94 ± 981.23a	0.0010
Organ relative weights3
Heart, %	0.52 ± 0.01a	0.47 ± 0.01b	0.0102
Liver, %	2.88 ± 0.26b	5.11 ± 0.25a	<0.0005
Spleen, %	0.11 ± 0.02a	0.06 ± 0.003b	0.0028
Abdominal fat, %	1.32 ± 0.05b	2.20 ± 0.12a	<0.0005
Bursa, %	0.19 ± 0.01a	0.07 ± 0.01b	<0.0005
Glycogen content3
Liver, mg/g	12.57 ± 1.41b	20.54 ± 2.41a	0.0127
Breast muscle, mg/g	1.19 ± 0.12b	2.50 ± 0.42a	0.0073
Leg muscle, mg/g	0.82 ± 0.03	0.84 ± 0.04	NS

¹Control, control group (basal diet); CORT, CORT-treated group (30 mg/kg diet); NS, nonsignificant.

²Values are expressed as mean ± SEM, n = 6.

³Values are expressed as mean ± SEM, n = 8.

^a,bMean values in a row sharing no common superscript are significantly different (P < 0.05).

Organ relative weights and tissue glycogen content

The effects of CORT on relative weights and tissue glycogen content in broilers are shown in Table 1. Compared with the control, CORT treatment decreased (P < 0.05) the relative weights of heart, spleen, and bursa and increased (P < 0.05) the relative weights of the liver and abdominal fat. The contents of glycogen in the liver and breast muscle of the chicks fed CORT were higher (P < 0.05) than those of chicks in the control group, but CORT had no significant effect on the glycogen content in the leg muscle of 14-d-old chicks (P > 0.1).

Appetite-related gene expression and AMPK signaling pathway in hypothalami

The effects of CORT on appetite-related gene expression and AMPK signaling pathway are shown in Figure 1. Compared with the control, the CORT treatment group increased (P < 0.05) the gene expression of mammalian target of rapamycin (mTOR), glucocorticoid receptor (GR), AMPKα2, neuropeptide Y (NPY), liver kinase B1 (LKB1), AMPKα1, and fatty acid synthase (FAS) in the hypothalami of 14-d-old broilers. However, CORT treatment had no effect on the gene expression of calmodulin-dependent kinase kinase α (CaMKKα) and calmodulin-dependent kinase kinase β (CaMKKβ) (P > 0.1). Compared with the control conditions, CORT treatment increased the protein levels of phosphorylated ACC, total AMPK, and phosphorylated AMPK in the hypothalami of 14-d-old broilers (P < 0.05). In contrast, the protein level of phosphorylated mTOR remained unchanged (P > 0.1).

Figure 1.

Effect of CORT treatment on the hypothalamic genes and proteins levels of AMPK signaling pathway in chicks. (A) Genes expression of CaMKKα, CaMKKβ, LKB1, AMPKα1, AMPKα2, NPY, GR, FAS, and mTOR in hypothalami. (B) Representative bands of hypothalamic proteins involved in the AMPK signaling pathway. (C) Proteins expression of phospho-ACC^Ser79, total AMPK, phospho-AMPKα ^Thr172, phospho-mTOR^Ser2448 in hypothalami. Control, control group (basal diet); CORT, CORT-treated group (30 mg/kg diet). ^a,bMeans (n = 8) sharing no common letters differ significantly (P < 0.05).

Discussion

Stress is the nonspecific response of the body to any demand (Hans, 1976) and refers to the systemic, nonspecific response upon exposure to strong stimulation or chronic stress (Yang et al., 2015), such as severe trauma, shock, infection, burn, major surgery, or improper environment (Du et al., 2019). These intense stimulatory factors may cause several nonspecific systemic reactions, including neuroendocrine responses and nerve-body fluid stress reactions, disturbing organisms, and damaging physical and psychological health (Yvonne and Karen, 2014).

Many studies focused on the effect of GCs on feed intake (FI) in broilers, and their results were different. Liu et al. (2012) found that CORT reduced FI and BWG in laying hens. A previous study has reported that feed intake was increased in chickens given higher doses of CORT but not in those given lower doses (Covasa and Forbes, 1995). In this study, CORT treatment did not affect the ADFI in 7- to 14-d-old broiler chickens, which was in agreement with previous studies (Lin et al., 2004a, 2004b; Yuan et al., 2008). The reason for no difference may be mainly attributed to the suppressed BW and limited the capacity of the gastrointestinal tract, which concealed the promotion of appetite induced by CORT. In addition, the feed intake caused by CORT administration in broilers was different and probably dependent on the dosage of CORT and the broiler diets. In the present study, ADG was remarkably reduced in CORT-treated broilers, which was consistent with previous findings (Lin et al., 2004a, 2004b; Yuan et al., 2008; Liu et al., 2012). The decrement in ADG may be attributed to the reduction in the absorption rate of nutrients in the small intestine (Hu et al., 2010) and proteolysis and gluconeogenesis induced by CORT (Lin et al., 2004a, 2004b).

Liu et al. (2012) found that peripheral CORT injection in laying hens increased CORT and GLU contents in plasma (Liu et al., 2012). In the present study, CORT treatment increased plasma CORT, GLU, TG, and TCHO contents. Song et al. (2011a) found that diet supplemented with 30 mg/kg CORT increased plasma GLU and TG. The increased CORT level indicated that the HPA system was activated in response to peripheral CORT treatment. The higher level of plasma CORT and simultaneous hyperglycemia indicated the induction of stress. Lin et al. (2004a, 2004b) found that in the case of long-term stress, the negative feedback regulation of GC in the HPA axis was impaired, thereby resulting in a continuous increase in GC secretion, lipid mobilization in adipose and muscle tissue, and the strengthening of glyconeogenesis in the liver, eventually leading to an increase in FFAs and GLU in the blood. An increase in plasma lipids and FFA is generally accepted as a characteristic manifestation of stress (Hans, 1976). Cai et al. (2009) found that increased hepatic de novo lipogenesis, and in turn, increased circulating lipid flux, contributes to augmented fat deposition in the adipose tissues and livers of dexamethasone-challenged chickens. Thus, in this study, the augmented fat deposition and elevated plasma lipid metabolites in CORT-challenged chickens indicated that hepatic de novo lipogenesis increased. All these results suggested that stress was successfully induced by CORT administration in the present study.

GCs mobilize substrates from peripheral energy depots such as adipose and skeletal muscle for use in hepatic gluconeogenesis, thereby ensuring a plentiful supply of GLU for use under conditions of stress (Liu et al., 2012). In this experiment, the contents of glycogen in the liver and breast muscle were increased by CORT, thereby indicating that under stress, the process of glyconeogenesis in the liver and breast muscle was strengthened. Similarly, GCs have been reported to induce muscular glycogen mobilization (Hazard et al., 2011), which results in the increased use of energy stores (Sapolsky et al., 2000) during stress response. Duan et al. (2014) found that CORT treatment significantly decreased the glycogen content in liver tissues, which was inconsistent with our study. The reasons may be due to the age and the different treatment of CORT. Plasma CORT could activate glycogen synthase (McMahon et al., 1988), thereby explaining the mechanism of the increased glycogen content caused by GCs in this study.

CORT increased energy consumption and altered energy redistribution toward fat deposition, inducing insulin resistance and enhancing de novo fatty acid synthesis in the liver, leading to fat deposition (Yuan et al., 2008). In this study, we found that CORT increased the amount of abdominal fat in chicks, which was consistent with the study of Yuan et al. (2008). CORT administration promoted energy mobilization from skeletal muscles and, in turn, augmented fat accumulation in either abdominal or subcutaneous adipose tissues (Yuan et al., 2008). In this study, CORT treatment reduced the relative weights of bursa and spleen indexes in broilers. GC could accelerate the degeneration of lymphoid tissues, such as the thymus and bursa of Fabricius, mainly due to lymphocyte DNA degradation (Compton et al., 1991). The mechanism of GC-induced apoptosis is still unclear and might be related to the cell type and the expression level of GR (Reichardt et al., 2000).

In poultry, the hypothalamus could integrate various factors in the external environment and make an appropriate response toward FI (Richards et al., 2010). The regulatory of GC on appetite was related to hypothalamic neurons (Liu et al., 2014). As in mammals, in the hypothalamus of avian species, two primary populations of neurons influence appetite through the release of signal molecules: one neuron population is composed of orexigenic neurons, which express NPY/agouti-related protein; the other neuron population is composed of anorexigenic neurons, which express proopiomelanocortin/cocaine- and amphetamine-regulated transcript, which is responsible for suppressing appetite. NPY is a highly conserved neuropeptide that plays roles in regulating energy homeostasis and appetite in mammals and poultry (Schwartz et al., 2000). GCs stimulated the expression of NPY in the ARC of the hypothalamus (Sato et al., 2005). In this study, CORT increased the expression of NPY mRNA. With stress factor-induced stimulation, the secretion of GC by the adrenal gland was increased, and CORT biological function was evident after binding to the GR (Covasa and Forbes, 1995). GR mRNA signals are widely distributed in the brain, such as in the nucleus rotundus, dorsolateral nucleus, nucleus ovoidalis, nucleus reticularis superior, hippocampus, paraventricular nucleus, periventricular nucleus, lateral hypothalamus, and ventromedial nucleus in broilers (Yuan et al., 2017). In the present study, dietary CORT increased the expression of GR mRNA in the hypothalamus, thereby indicating that the biological regulation of GC was strengthened.

LKB1 and CaMKKβ are kinases upstream of AMPK, and research has found that LKB1 exhibits the same function both in poultry and in mammals (Proszkowiec-weglarz et al., 2006). Unlike LKB1, whose expression is ubiquitous, the expression levels of CaMKKα and CaMKKβ are more restricted; high expression levels were found in the brain, including the hypothalamus, whereas low expression levels were found in peripheral tissues (Vinet et al., 2003). In the present study, CORT increased LKB1 expression but had no effect on the expression of CaMKKα and CaMKKβ, thereby suggesting that the activation of AMPK was mainly due to the upstream kinase LKB1. In addition, CORT upregulated the mRNA expression of AMPKα 1 and AMPKα 2 and increased the protein levels of total AMPK and phosphorylated AMPK in this study, thereby suggesting that CORT ultimately resulted in the activation of AMPK in the hypothalamus. Notably, alterations in AMPK in this pattern may decrease anabolism and/or increase catabolism (Xue and Kahn, 2006), which was also reflected in the changes in carbohydrate and lipid metabolism observed in the present study. Moreover, CORT treatment increased the protein level of phosphorylated ACC, which is consistent with the results of previous studies (Liu et al., 2014). The activation of AMPK and consequent inhibition of ACC inhibited lipogenesis and increased fatty acid oxidation (Kola et al., 2006).

In conclusion, the results of the present investigation indicated that dietary CORT activated the LKB1-AMPK-NPY/ACC signaling pathway of the hypothalamus to regulate appetite-related gene expression and energy balance. In general, the central AMPK signaling pathway is essential to regulating various signals related to energy balance, especially under stress. These findings could provide a mechanism for explaining how stress could affect appetite-related genes and energy metabolism.

Glossary

Abbreviations

ACC

acetyl-coenzyme A carboxylase

ADFI

average daily feed intake

AMPK

adenosine 5′-monophosphate-activated protein kinase

AMPKα1

AMP-activated protein kinase α1

AMPKα2

AMP-activated protein kinase α2

ARC

arcuate nucleus

BW

body weight

BWG

body weight gain

CaMKKα

calmodulin-dependent kinase kinase α

CaMKKβ

calmodulin-dependent kinase kinase β

cDNA

complementary DNA

CNS

central nervous system

CORT

corticosterone

CT

cycle threshold

ADG

average daily gain

F:G

feed-to-gain ratio

FAS

fatty acid synthase

FFA

free fatty acid

FI

feed intake

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GC

glucocorticoid

GLU

glucose

GR

glucocorticoid receptor

HPA

hypothalamus–pituitary–adrenal

mTOR

mammalian target of rapamycin

NPY

neuropeptide Y

LKB1

liver kinase B1

PCR

polymerase chain reaction

RT

reverse transcription

SYBR

synergy brands

TCHO

total cholesterol

TG

triglyceride

Conflicts of interest statement

The authors declare no conflict of interest.