1. Introduction
In recent years, affected by the international trade situation, corn import volume and import price have risen sharply in China, which has increased the production cost of poultry farming [
1]. Therefore, poultry nutritionists have been encouraged to use alternative grains instead of corn. Grains mainly supply starch and serve as the primary source of energy. Starch includes two main components: amylose (
AM) and amylopectin (
AP). Previous reports have demonstrated that AP is easier to digest than AM [
2], and starches with a higher ratio of AP may lead to a sharp rise in blood glucose and insulin levels [
3]. Additionally, researchers found that the digestion kinetics of starch and amino acids are interdependent, with the properties of starch digestion affecting the metabolic pathways of amino acids [
4]. When rapidly digestible starch (
RDS, a low AM/AP starch) is consumed, glucose is rapidly released in the small intestine, a process that may not be able to continuously meet the normal energy requirements of broilers. In such cases, more amino acids may be oxidized to provide the energy needed by the body, resulting in a decrease in amino acid utilization [
5].
Lysine (
Lys) is defined as a basic essential amino acid whose carbon skeleton cannot be synthesized by poultry and therefore must be met through the diet for the maintenance and growth of poultry [
6]. Lys is the second-most-limiting amino acid after methionine in corn–soybean meal-based diets for broilers. It is considered to be one of the main essential amino acids for muscle growth, development and deposition in broilers [
7], especially for the turnover of breast muscle protein to regulate protein biosynthesis and decomposition [
8,
9]. At present, researchers have reported inconsistent results regarding the effect of dietary Lys on abdominal fat deposition in broilers. Maqsood et al. [
10] believed that increasing dietary Lys levels could reduce the generation of abdominal fat in broilers. Tian et al. [
11] found that a lack of dietary Lys can lead to a reduction in abdominal fat in broilers. However, the underlying mechanism by which dietary Lys directly or indirectly regulates breast protein and liver lipid metabolism in broilers is still unclear.
The synthesis of new muscle proteins is highly regulated by multiple signals integrated by the mammalian target of rapamycin (
mTOR) [
12]. In fact,
mTOR promotes the synthesis of new proteins by directly phosphorylating the expression of translation-related downstream proteins, such as the signaling molecules ribosomal protein S6 kinase 1 (
S6K1) and eukaryotic initiation factor 4E binding protein-1 (
Eif4E) [
13,
14]. In addition, muscle protein content is also controlled by protein degradation, and protein degradation pathways are mainly divided into the ubiquitin–proteasome pathway (
UPP) and autophagy–lysosome pathway [
15]. The UPP is a critical protein breakdown pathway in eukaryotes, and scientists have demonstrated its importance in the muscle degradation process [
16].
The liver is the center of glycogen synthesis, gluconeogenesis and energy metabolism in poultry. It acts as a central regulator of lipid homeostasis and is responsible for coordinating the synthesis, export and utilization of fatty acids as energy substrates [
17,
18]. Lipid metabolism in the body is mainly regulated by the adenosine 5`-monophosphate-activated protein kinase (
AMPK) signaling pathway. The activation of
AMPK inhibits lipogenesis and deposition while increasing fatty acid oxidation, affecting cholesterol and triglyceride synthesis, thereby regulating cellular energy balance [
19]. A prior study noted that some genes such as peroxisome-proliferator-activated receptor α (
PPARα), carbohydrate-responsive element-binding protein (
ChREBP), sterol regulatory element-binding protein-1c (
SREBP-1c), malic enzyme (
ME) and acetyl CoA carboxylase (
ACC) participate in hepatic lipid metabolism [
11,
20,
21].
It is unclear whether feeding broiler chickens with different dietary starch sources would affect the utilization of dLys in their bodies, thereby affecting broiler breast muscle protein and liver lipid metabolism, due to differences in the digestion rates of these different starch sources. Therefore, the purpose of this study was to investigate the effects of different dLys levels on broiler carcass traits, serum metabolites and postprandial glucose and insulin changes under different dietary starch sources and to explore the responses of genes related to breast muscle protein and liver lipid metabolism to different starch sources and different dLys levels.
2. Materials and Methods
2.1. Ethics Statement
All animal procedures were conducted in accordance with the Beijing Regulations of Laboratory Animals (Beijing, China) and were approved by the Laboratory Animal Ethical Committee of China Agricultural University (Protocol Number: AW03602202-1-3).
2.2. Experimental Diets and Treatments
A total of 702 one-day-old male Arbor Acres Plus broiler chickens (from Beijing Poultry Breeding Company, Beijing, China) were randomly divided into 9 treatment groups based on a 3 × 3 two-factor experimental design. The treatments consisted of 3 different starch sources (corn, cassava and waxy corn) with 3 different dLys levels (1.08%, 1.20% and 1.32%). Each group included 6 replicate cages with 13 birds each. The experimental period was 21 days. The formula and nutritional level of the experimental diets are shown in
Table 1.
2.3. Bird Husbandry
Bird management was based on the guide for Arbor Acres Plus broilers. All birds had access to feed and water ad libitum in crumble-pellet form and via nipple drinkers, respectively.
2.4. Sampling Procedures
On day 19, the birds from the dietary 1.20% dLys groups of different starch sources and the cassava starch groups of different dLys levels were selected. After fasting for 4 h, feeding for 0.5 h (set as zero) and at 0.5, 1.0, 1.5 and 2.0 h after feeding, blood samples were collected from birds’ wing veins in each group and kept in sterile serum collection tubes. Blood was collected once from each bird to avoid the influence of stress on the experimental result.
On day 21, six birds close to the average weight (one bird/replicate) in each treatment were selected for sample collection. The blood samples were drawn from birds’ wing veins and kept in sterile serum collection tubes. The birds were euthanized after intravenous injection of sodium pentobarbital (30 mg/kg). Six small pieces of the pectoralis and liver samples were washed with saline solution and collected into 1.5mL sterile Eppendorf tubes, and then snap-frozen in liquid nitrogen immediately and stored at −80 ℃ for mRNA analysis.
2.5. Carcass Characteristics Determination
On day 21, six birds close to the average weight (one bird/replicate) in each treatment were selected for slaughter, after which the breast muscle (%) and abdominal fat (%) were determined.
2.6. Blood Metabolite Analysis
The collected blood samples were centrifuged at 5000× g at 4 °C for 10 min, and the obtained serum samples were stored at −80 °C for later testing of glucose, cholesterol, triglycerides, uric acid and blood urea nitrogen. The serum glucose, insulin, urea nitrogen, uric acid, total cholesterol and triglycerides were analyzed using an XH-6080 radioimmunoassay analyzer.
2.7. Quantitative Real-Time PCR Analysis
The total RNA was extracted from the pectoralis and liver tissues using an RNAiso plus (Takara, Kyoto, Japan) according to the manufacturer’s recommendations. The nucleic acid concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA purity was verified using 1.5% denaturing agarose gels. Next, cDNA was synthesized by using a high-capacity cDNA reverse transcription kit (Takara, Kyoto, Japan) and stored at −20 °C. All quantitative real-time PCR (qRT-PCR) assays were performed using an SYBR Premix ExTap kit (Takara, Kyoto, Japan) in a 7500-fluorescence detection system (Applied Biosystems, Carlsbad, CA, USA). The primer sequences for qRT-PCR are listed in
Table 2. All gene values were normalized to the expression of the housekeeping gene β-actin.
2.8. Statistical Analysis
The data were first tested for homogeneity of variances, after which the general linear model (
GLM) of SPSS 20.0 statistical software (version 20.0, SPSS Inc., Chicago, IL, USA) was used to conduct a two-factor analysis of variance. The statistical model for this study is shown below. Differences between the treatment groups were considered statistically different at
p < 0.05. All experimental data (carcass traits, serum metabolites, breast muscle protein metabolism and liver lipid metabolism) were based on one bird in each cage as the experimental unit. The different starch sources and different levels of digestible lysine were used as independent variables. The carcass characteristics, blood biochemistry, breast muscle protein and liver metabolic genes were used as dependent variables.
Here, Yijk is the kth observation of the dependent variable recorded on the ith and jth treatments, μ is the overall mean, αi is the effect of the ith treatment, βj is the effect of the jth treatment, (α × β)ij is the interaction effect of the ith and jth treatments and εijk is the error associated with Yijk.
4. Discussion
Previous research found that Lys not only affected the process of body protein deposition and promoted the development of muscles (being especially important for the development of pectoral muscles) [
22], but also regulated the expression of lipid synthesis genes and affected the deposition of fat in the body [
11]. However, an unexpected result in this study was that dLys levels had no significant effect on broiler breast muscle growth. It is generally believed that broiler breast muscle growth increases with increasing Lys levels in the diet. Tesseraud et al. [
9] proposed that Lys deficiency reduced muscle weights and protein contents by approximately −50% and −60% for the breast muscle and approximately −25% for the sartorius muscle. Tian et al. [
11] found that broiler breast muscle growth with a Lys level of 0.60% in the diet was far lower than that of broilers with Lys levels of 1.00% and 1.40%. Based on these reports, we guessed that the insignificant results caused by adding different dLys levels in this study may be because the differences in dLys levels were too small and the broilers did not reach a state of deficiency or excess. Abdominal fat is an important indicator of fat deposition in broilers; excessive fat deposition is undesirable as it reduces feed efficiency and meat quality and increases production and health costs [
23,
24,
25]. Previous entries in the literature have reported that higher Lys levels in the diet could help reduce the percentage of abdominal fat in broilers [
10]. Similarly, this study also found that increasing dLys levels in the diet could reduce the percentage of abdominal fats of broilers fed with different dietary starch sources. To a certain extent, this shows that increasing dLys levels could reduce the waste of nutrients in the body, thereby promoting their distribution in the development of other tissues and organs, but the specific distribution method remains to be clarified by subsequent studies. In addition, this study also found that the percentage of abdominal fat in broilers in the waxy corn starch diet group was significantly higher than that of the other two groups. This is because a low AM/AP ratio diet induces faster and stronger blood glucose and insulin responses, resulting in the upregulation of genes involved in hepatic lipid synthesis and the increased activity of lipogenic enzymes [
26,
27], ultimately leading to abdominal fat deposition. In summary, we recommend that the dLys level in the diet should be appropriately increased when the waxy corn starch diet is used.
Uric acid is the end product of amino acid catabolism, and any amino acid not required as a building block during protein synthesis is broken down. During amino acid catabolism, the amino terminus of each amino acid must be removed. Because nitrogen compounds are not utilized in energy transduction pathways, the accumulation of amino groups may have adverse effects on the animal. Currently, researchers believe that serum uric acid levels can be used as a measure of amino acid availability [
28] and, because uric acid levels in serum correlate with urinary and fecal nitrogen excretion rates, as a measure of nitrogen utilization. The formation of uric acid in the blood affects the efficiency of protein deposition in the muscles. In this study, it was found that, except for serum cholesterol, other serum metabolites were not affected by the dLys levels, which is consistent with the findings of Zarghi et al. [
29]. At the same time, this is also consistent with the results of breast muscle growth in this study, because the breast muscle growth of each treatment group was not affected by the dLys levels. Cholesterol can be obtained from feed or synthesized in the body, and the synthesis rate of cholesterol is related to the lipid metabolism rate in the body. Studies by Emadi et al. [
30] have shown that amino acid supplementation in diets could reduce blood cholesterol levels in broilers. This is consistent with the results of this study, where we observed that adding 1.32% dLys to broiler diets significantly reduced their serum cholesterol levels. This is also consistent with the results of abdominal fat production rates in this study, which demonstrate that the serum cholesterol level was positively correlated with the percentage of abdominal fat.
The amount of native starch hydrolyzed by amylase in the animal gut is negatively correlated with the dietary AM content [
31]. Currently, high AM has been shown to differ from high AP in postprandial serum glucose and insulin responses and is associated with a lower rate of amylolysis [
32,
33]. The same phenomenon was also observed in the present study, where we determined the effects of corn, cassava and waxy corn starch diets on serum glucose and insulin changes in broilers within 2 h after feeding. As expected, the cassava and waxy corn starch diets contained higher levels of AP, which promoted faster and greater serum glucose and insulin responses in the body. This is in the same trend as the results of Ma et al. [
34], and researchers have also observed similar phenomena in mammals [
35,
36]. In addition, we also observed that increasing dLys levels in the diet helped maintain the stability of glucose in the body. This may be because there is a certain interaction effect between higher dLys levels in the diet and glucose in the intestine, thereby promoting the continuous transfer of glucose in the intestine to the blood.
Amino acids play a key role in regulating protein synthesis or degradation in various tissues, including skeletal muscle. A study by Watanabe et al. [
37] confirmed that feeding a low-Lys diet can significantly increase the mRNA expression of protein-degradation-related genes in broiler muscle. Li et al. [
38] found that leucine stimulated
mTOR signal passage and affected skeletal muscle protein synthesis. Similarly, Zheng et al. [
39] also found that leucine could activate the
mTOR signaling pathway, thereby enhancing the phosphorylation expression of its downstream signaling molecules
S6K1 and
Eif4E and finally promoting protein synthesis in dairy cow skeletal muscle.
S6K1 mainly accelerates protein synthesis by enhancing translation initiation factors [
40], while
Eif4E promotes protein synthesis by stimulating the translation of a subset of mRNAs with 5`-terminal pyrimidine motifs [
41]. Therefore, we speculated that the increase in muscle protein synthesis caused by Lys is also mediated by enhancing the translation initiation of its related mRNAs [
42]. In the current study, we found that increasing the levels of dLys in the diet could significantly upregulate the expression of protein-synthesis-related genes such as
mTOR,
S6K1 and
Eif4E, which confirmed our speculation that increases in muscle protein synthesis caused by Lys are also mediated by enhancing the translation initiation of its related mRNAs. In addition, this study also found that a 1.32% dLys level could down-regulate the expression of protein-degradation-related genes such as muscle RING finger protein (
MuRF) and muscle atrophy factor 1 (
Atrogin-1).
Atrogin-1, muscle atrophy F-box protein (
MAFbx) and
MuRF are the most representative E3 ubiquitin ligases in skeletal muscle, which mediate the polyubiquitination of proteins and target them to the 26S proteasome for degradation [
43]. Currently, the expression of
MAFbx,
MuRF and
Atrogin-1 has been found to be significantly upregulated in various conditions that lead to muscle degradation [
44]. Although there were no differences in breast muscle deposition in the current study, it increased numerically with increasing dLys levels. This further illustrated that Lys caused the enhancement of protein deposition in broiler muscle by promoting protein synthesis and inhibiting protein degradation.
As the main transcription factors in the
AMPK signaling pathway,
PPARα and
SREBP-1c have been shown to play key roles in liver lipid metabolism [
45]. In this study, we found that increasing dietary dLys levels significantly upregulated the mRNA expression of
AMPK and
PPARα in the liver tissue of broilers.
PPARα activation induces the transcriptional upregulation of fatty acid transporters and downregulation of fatty acid synthesis [
46]. Similarly, Sugden et al. [
47] also showed that
PPARα enhanced fatty acid β-oxidation by regulating the expression of
PPARα target genes, thereby reducing the accumulation of lipids in vivo. In summary, increasing dietary dLys levels could activate the
AMPK signaling pathway, thereby promoting the transcription level of
PPARα, and finally inhibiting the accumulation of abdominal fat in broilers. Furthermore, the intake of a high AP diet can rapidly elevate blood glucose content in broilers, and blood glucose can change the expression of related transcription factors in a series of tissues through the lipid synthesis pathway, thus directly affecting the expression of lipogenic genes [
27]. In the present study, the waxy corn starch diet significantly upregulated the expression of
AMPK,
CPT1,
FABP1 and
ACC transcription factors. Although the genes related to lipid synthesis and decomposition were significantly upregulated in the broilers fed with the waxy corn starch diet, combined with the previous phenotypic results, we judge that the rate of lipid synthesis was significantly higher than the rate of lipid decomposition, which eventually led to abdominal fat deposition in the broilers. Our findings further support the results of Yang et al. [
27] and Yin et al. [
36].