CAMKK2-AMPK axis endows dietary calcium and phosphorus levels with regulatory effects on lipid metabolism in weaned piglets

CAMKK2-AMPK axis endows dietary calcium and phosphorus levels with regulatory effects on lipid metabolism in weaned piglets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CAMKK2-AMPK axis endows dietary calcium and phosphorus levels with regulatory effects on lipid metabolism in weaned piglets zhenyan miao, Yanjie Sun, Zhangjian Feng, Qiwen Wu, Xuefen Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4069024/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted 5 You are reading this latest preprint version Abstract Background In the realm of swine production, optimizing body composition and reducing excessive fat accumulation is critical for enhancing both economic efficiency and meat quality. Despite the acknowledged impact of dietary calcium (Ca) and phosphorus (P) on lipid metabolism, the precise mechanisms behind their synergistic effects on fat metabolism remain elusive. Results Research observations have shown a decreasing trend in the percentage of crude fat in carcasses with increased calcium and phosphorus content in feed. Concurrently, serum glucose concentrations significantly decreased, though differences in other lipid metabolism-related indicators were not significant across groups. Under conditions of low calcium and phosphorus, there is a significant suppression in the expression of FABPs, CD36 and PPARγ in the jejunum and ileum, leading to inhibited intestinal lipid absorption. Concurrently, this results in a marked increase in lipid accumulation in the liver. Conversely, higher levels of dietary calcium and phosphorus promoted intestinal lipid absorption and reduced liver lipid accumulation, with these changes being facilitated through the activation of the CAMKK2/AMPK signaling pathway by high-calcium-phosphorus diets. Additionally, the levels of calcium and phosphorus in the diet significantly altered the composition of liver lipids and the gut microbiota, increasing α-diversity and affecting the abundance of specific bacterial families related to lipid metabolism. Conclusion The evidence we provide indicates that the levels of calcium and phosphorus in the diet alter body fat content and lipid metabolism by modulating the response of the gut-liver axis to lipids. These effects are closely associated with the activation of the CAMKK2/AMPK signaling pathway. Dietary calcium and phosphorus Lipid metabolism Intestine-liver axis CAMKK2 AMPK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Calcium and phosphorus, fundamental to the mineral nutrient spectrum, play an indispensable role in bone development and act as critical secondary messengers in cellular signaling. The significance of these minerals transcends the mere provision of skeletal integrity, extending to a broad spectrum of physiological processes, including muscle contraction, the facilitation of neurotransmitter dissemination, hormone secretion, and the regulation of body weight equilibrium. These roles underscore the indispensable contribution of calcium and phosphorus to both developmental and homeostatic mechanisms within the biological system[1; 2]. In the field of lipid metabolism, calcium and phosphorus are essential for the maintenance of a healthy state. Calcium augments energy utilization and weight regulation through the facilitation of lipolysis, amplification of insulin sensitivity, meticulous regulation of fatty acid oxidation, and nuanced modulation of cholesterol levels. On the other hand, phosphorus occupies a central position in energy metabolism. The role of phosphorus in ATP production is well-established, significantly impacting lipid synthesis and catabolism, lipoprotein metabolism, and, through the regulation of hormones like insulin, subsequently influencing the overarching lipid equilibrium. Calcium supplementation has been shown to mitigate organ fat accumulation induced by a high-fat diet and to enhance intramuscular fat storage in livestock, indicating its potential in improving meat quality and animal health[ 3 ]. Conversely, whilst phosphorus supplementation was able to reduce overall lipid content, it also reduced intramuscular fat content, demonstrating its subtle effects on lipid distribution within muscle tissue[1; 4]. Previous studies have focused on the dual regulation of calcium and phosphorus on muscle performance, leaving a gap in understanding the effects of dietary calcium and phosphorus changes on overall lipid metabolism and the mechanisms involved. This knowledge gap underscores the need for a more comprehensive exploration of how these minerals interact in complex networks of lipid metabolism that may provide new nutritional strategies for animal health and production performance. In the animal body, lipids play a multifaceted and critical role, not only as a major energy reservoir, but also influencing livestock production and meat quality. However, excessive accumulation of fat not only reduces lean meat percentage and meat quality, but also affects feed conversion efficiency and may even lead to serious health problems such as fatty liver[ 5 ]. In animals, fats are primarily synthesized through the esterification of fatty acids with glycerol, serving as the main source of energy reserves in the body. [ 6 ]. Lipids not only serve as the energy base necessary for the maintenance of daily physiological activities, but also play key roles in a variety of biological processes, including thermoregulation, construction and maintenance of cell membrane structure and function, regulation of insulin sensitivity, protection of vascular health, and participation in immune responses[ 6 ]. In addition, adipose tissue is an organ with important endocrine functions. It plays a key role in the regulation of metabolic health and energy balance through the secretion of a variety of bioactive substances, such as lipocalins, resistin and leptin. Therefore, understanding and controlling fat accumulation and its metabolic processes are important for improving meat quality, enhancing animal health, and improving feed efficiency. As a central regulator of intracellular energy metabolism, 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is essential for maintaining the stable availability of glucose, glycogen and fatty acids. In addition, AMPK plays a key role in signaling pathways that sense intracellular lysosomal and nuclear DNA damage[ 7 ]. Studies have shown that AMPK has an important role in the regulation of lipid and glucose metabolism in the heart, hypothalamus, adipose tissue, muscle, and liver. Calcium has a marked inhibitory effect on endogenous lipid production in the liver through activation of the AMPK pathway, but the deeper molecular mechanisms have not been elucidated[ 8 ]. As an upstream signal of AMPK, calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) can enhance its regulatory effect on lipid metabolism by activating AMPK, and conversely AMPK activation can influence Ca²⁺ signaling and modulate CaMKK2 activity. The function of CaMKK2, especially in regulating lipid metabolism has been demonstrated in several studies. Both genetic deletion and pharmacological inhibition of CaMKK2 significantly reduces de novo fatty acid synthesis and may bring about amelioration of high-fat diet-induced fatty liver, reduced insulin sensitivity, and prostate cancer cell proliferation[9; 10]. Although calcium and phosphorus, as key mineral nutrients, play important roles in maintaining the body's lipid metabolism homeostasis, the understanding of how different dietary calcium and phosphorus intakes specifically affect lipid metabolism and their molecular mechanisms is incomplete. In view of this, the aim of this study was to investigate the effects of different calcium and phosphorus levels (normal, low and high) on lipid metabolism and the mechanisms behind them using a weaned piglet model. In particular, this study focused on the interaction between intestine and liver during lipid absorption and processing, revealing how increased calcium and phosphorus levels ameliorate disordered lipid metabolism by activating the AMPK/CAMKK2 pathway in the intestine-liver axis. Further, this study also examined the correlation between gut microbiota and liver lipid composition, exploring how the intestine-liver axis serves as a key mechanism by which dietary calcium and phosphorus regulate lipid metabolism. This comprehensive study not only deepens our understanding of the role of calcium and phosphorus in the regulation of lipid metabolism, but also provides new insights into how to optimize lipid metabolism by modulating calcium and phosphorus intake in the diet. 2 Materials and methods 2.1 Ethics Statement All animal procedures were carried out in accordance with the guidelines for the care and use of experimental animals of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences, and approved by the Animal Care Advisory Committee of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences. 2.2 Animals and experimental protocol Before the study began, all the components of Ca and P used to produce the experimental diet were measured in the laboratory, the standardized total tract digestible (STTD) Ca and STTD P values of feeds were calculated based on the STTD Ca and STTD P data of feedstuffs for further formulation of feed formulations. The basal ration was a corn-soybean meal type ration, the ratio of dietary STTD Ca to STTDP was fixed at 1.2, STTD Ca varied with STTD P, and other nutrient levels were formulated with reference to NRC(2012), and the composition of the diets is shown in Table S1 . In order to eliminate the effect of phytase, phytase was not added in the experiment. Seventy-two weaned piglets ( Duroc × Landrace × Yorkshire , 25 days of age) with an initial body weight (BW) of 7.23 ± 0.92 kg were randomly assigned to three treatment groups, with six replicate pens of four weaned piglets per treatment. Control (CON) pigs were fed a diet with an STTD Ca of 0.504 and an STTD P of 0.42. Pigs in the low calcium-phosphorus group (LCAP) were fed a diet with an STTD Ca of 0.216 and an STTD P of 0.18. Pigs in the high calcium-phosphorus group (HCAP) were fed a diet with an STTD Ca of 0.696 and an STTD P of 0.58. Animals are fed in separate enclosures (of the same size). All pigs are fed three times a day and are given clean water free of charge. The experiment lasted six weeks. Food intake was recorded daily and initial and final weight was measured at the beginning and end of the experiment, respectively. 2.3 Sample collection Prior to slaughter, blood samples were collected and centrifuged for 12 min (3000rpm/min, 4°C) to obtain serum, which was stored at -4°C for further determination. The intestinal, muscle, chyme and liver tissues were rapidly frozen in liquid nitrogen immediately after slaughter and stored at -80°C for further study. A portion of liver, intestine and muscle tissues were fixed in 4% paraformaldehyde for histological observation. 2.4 Carcass chemical analysis First, the whole carcass (n = 6) including the bones of the three groups of piglets were frozen and processed into bone paste by using different sizes of grinders. The chopped samples were thoroughly homogenized in a blender. Samples (500 g) were then collected, vacuum packed and stored at -20°C until further analysis. The total fat content of carcass was determined by Soxhlet extraction method. Briefly, three replicates of each sample were taken and about 30 g of carcass samples were ground into minced meat, dried in a vacuum freeze dryer and then ground into powder. One g of dried meat sample (accurate to 0.0001 g) was wrapped in filter paper into a cylindrical filter cup and extracted with n-hexane at 140°C for 50 min in a Soxhlet extraction unit (SE-A6, alva, Jinan, China). After air-drying for 10 min and baking at 102°C for 30 min, the extracted oil-containing aluminium cups were accurately weighed (to the nearest 0.0001 g) when the aluminium cups were cooled to room temperature. Total carcass fat (%) = (weight of oil-containing aluminium cups after extraction - weight of empty aluminium cups)/weight of air-dried meat samples × 100. 2.5 Biochemical analysis Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) concentrations, and glucose (GLU) concentrations were determined using an automated biochemical analyser (ICUBIO, iMagic-M7, Shenzhen, China). Intestinal samples, liver samples, muscle samples and carcass samples were homogenised in saline solution (1:9) and sediment was removed by centrifugation (3000 rpm, 10 min) to obtain 10% tissue homogenate. The TC and TG levels in the intestines, liver, muscles, and torso were all measured using colorimetric assay kits(Nanjing Jiancheng Biotechnology Company, Nanjing, China) according to the manufacturer's instructions. 2.6 Histological analysis The liver tissues of piglets fixed in 4% paraformaldehyde were frozen and embedded. The cryosections were cut into 10 µm thickness and stained with Oil red O. The sections were washed with 85% propylene glycol and then with distilled water before being stained with hematoxylin. The staining process was repeated after each wash. The presence of lipid droplets was indicated by a red stain. Sections were examined under a microscope (Nikon, Tokyo, Japan), photographed and recorded using Image-Pro Plus 6.0 software for subsequent comparison. 2.7 Real-time quantitative PCR analysis Total RNA from tissues was extracted with TRIzol reagent (Takara Bio, Shiga, Japan). 1µg of RNA was then reverse transcribed to cDNA using the PrimeScript RT kit with cDNA Eraser (Takara Bio) according to the kit instructions. 1µg of RNA was extracted from the tissue or cells with the fluorescence quantification kit SYBR® Premix Ex Taq (TaKaRa Bio) was used to perform real-time PCR on the CFX Connect Detection System (Bio-Rad, Hercules, CA, USA).The 20µL of reaction system showed as below, SYBR Premix Ex Taq (10µL), upstream primer (0.4µL), upstream primer (0.4µL), downstream primer (0.4µL), ROX II dye (0.4µL), cDNA samples (2µL), and ultrapure water (6.8µL). The real-time quantitative PCR reaction conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 5 s, annealing at 60°C for 34 s, extension at 95°C for 15 s, and the number of cycles of amplification was 40. The results were calculated by the 2 −ΔΔCt method, and all the primer sequences required for qPCR in this study were shown in Table S2 . 2.8 Western blot analysis The samples were separated by SDS-PAGE and the protein was transferred to PVDF membrane. The following antibodies were used in western blotting, such as β-actin (Affinity, T0022, 1:3000), PPARγ (Abcam, 209350, 1:1000), SIRT1 (Cell Signaling Technology, 9475S, 1:1000), AMPKα (Cell Signaling Technology, #5831, 1:1000), phospho-AMPKα (Thr172) (Cell Signaling Technology, #2535, 1:1000), CAMKK2 (Proteintech, 111549-1-AP, 1:1000), phospho-CaMKK2 (Ser511) (Cell Signaling Technology, #12818, 1:1000), DGAT1(Abcam,ab54037, 1:1000),ACC1(Cell Signaling Technology, #4190, 1:1000), SREPB1C(Abcam, ab28481, 1:1000), and FASN (Cell Signaling Technology, 3180S, 1:1000). 2.9 Immunofluorescence staining Fixed intestinal tissues were embedded in paraffin and cut into 4µm sections, then dewaxed, rehydrated, and treated in microwave oven with EDTA-containing antigen retrieval buffer (PH 8.0). Afterwards, sections were blocked with 5% fetal bovine serum (Bioss, Beijing, China) for 1 h at room temperature and then incubated overnight (1:500 dilution) at 4°C with rabbit anti-AMPKα, CD36, FABP4 (Service Bio, Wuhan, China). After rinsing with PBS, they were incubated with Alexa Fluor 555(BBI, Shanghai, China)-conjugated goat anti-rabbit secondary antibody for 30 min at room temperature in the dark. In addition, images were obtained under a microscope with magnification of 200 (Nikon, Tokyo, Japan) and positive results were quantified using ImageJ software. 2.10 Non-targeted lipidomics of liver The total lipids were extracted from the livers of three groups of piglets. After thawing, liver tissue (20 Mg) was homogenized in 1 ml mixture (methanol, MTBE, and internal standard mixture). After that, ultrasound was performed at 4°C for 20 minutes and then left at room temperature for 30 minutes. The solution was centrifuged at 10°C at 14000 g for 15 min to obtain an upper organic solvent layer and dried under nitrogen. Samples were analyzed using a high-performance liquid chromatography system (UHPLC Nexera Shimadzu LC-30A) and LC separation was performed on a Waters ACQUITY PREMIERCSH C18 column (1.7µm, 2.1 mm × 100 mm). The lipid extract was redissolved in 200 mL of 90% isopropanol/acetonitrile, centrifuged at 14000 g for 15 min, and finally injected with 3 mL of the sample. Solvent A was acetonitrile-water (6:4, v/V) containing 0.1% formic acid and 0.1 mM ammonium formate, and solvent B was acetonitrile-isopropanol (1:9, v/V) containing 0.1% formic acid and 0.1 mM ammonium formate. The initial flow rate was 300µL/min with 40% Solvent B. Hold for 3.5 min, then linearly increase to 75% of solvent B within 9.5 min, then linearly increase to 99% of solvent B within 6 min, then equilibrate in 40% of solvent B for 5 min. The mass spectra were obtained by Q-exactive Plus in both positive and negative modes. All measured ESI parameters were optimized and preset as follows: source temperature, 300°C; capillary temperature, 350°C, ion spray voltage set to 3000 V, and S-Lens RF level set to 50%, respectively, the scanning range of the instrument is set to M/z 200–1800. Lipid search was used for peak recognition, peak extraction, and lipid identification (secondary identification). Fatty acid composition expressed as a percentage of total fatty acids. 2.11 16S rRNA sequencing and processing Total genomic DNA of the gut microbiota was extracted from colon contents samples using a DNA isolation kit (Omega Bio-Tek, Norcross, GA, USA). DNA concentration was determined using a Nanodrop instrument (Thermo Fisher Scientific). DNA integrity was assessed using 2% agar gel electrophoresis. Universal forward primer (5'-CCTAYGGGGRBGCASCAG-3') and reverse primer (5'-GGACTACNNGGGGTATCTAAT − 3') were used to amplify the V3-V4 region of the bacterial 16S rRNA gene. Sequencing was performed on the Illumina MiSeq/NovaSeq platform. The DADA2 module in QIIME2 (Version QIIME2-202202) software was used for noise reduction. Microbial composition diversity was analysed using QIIME and R software. PICRUSt2 software was used for KEGG pathway analysis. 2.12 Short-chain fatty acid analysis Short-chain fatty acids in colon contents were determined using gas chromatography. To prepare the sample, 50 mg of colon contents were mixed with 250 µL of ultrapure water for 5 minutes. The suspension was then centrifuged at 5000 rpm for 30 minutes. Next, 1 mL of supernatant was transferred to a 2 mL PE tube and mixed with 200µL of 42 mmol/L crotonic acid and 200µL of 10% metaphosphoric acid solution. The PE tubes were refrigerated overnight at 4°C and then centrifuged at 10,000 rpm for 10 minutes at 4°C. The resulting supernatant was mixed with an equal amount of ether and extracted for 5 minutes. The ether layer was aspirated using a disposable syringe, filtered through a 0.22µm organic membrane, and then injected into a brown vial for injection. Short-chain fatty acids (acetic, propionic, butyric, valeric, isobutyric and isovaleric acids, with crotonic acid as an internal standard) were measured using a gas chromatograph and a mass spectrometry detector (7890A and 5975C Inert XL EI/CI Mass Detectors, Agilent Technologies, Santa Clara, CA, USA).Detection of short-chain fatty acids was performed according to a previous GC procedure[ 11 ]. 2.13 Data analysis The results were statistically analyzed using SPSS 21.0 statistical analysis software, and the data were expressed as mean ± standard deviation. Statistical treatment was performed using one-way analysis of variance (ANOVA) followed by LSD post-test. p < 0.05 was considered statistically significant. 3 Results 3.1 Effect of calcium and phosphorus content on lipid homeostasis In a six-week dietary intervention study (Fig. 1 A), we systematically evaluated the effects of dietary calcium and phosphorus on lipid homeostasis in piglets. We first measured the percentage of crude fat in the carcass and the level of total triglyceride to assess the direct effect of dietary calcium and phosphorus on lipid accumulation. As shown in Fig. 1 B, the percentage of crude fat in carcasses showed a decreasing trend as the calcium and phosphorus content of the feed increased. In addition, the measurements of total triglyceride levels in carcasses (Fig. 1 C) were consistent with the observations of crude fat percentage, suggesting that lipid accumulation was regulated by feed calcium and phosphorus content. Further analysis of serum biochemical parameters of piglets (Fig. 1 D) revealed that in piglets fed with high calcium and phosphorus diets, the serum glucose concentration was significantly lower than that of CON group and LCAP group ( P < 0.05). In addition, the ratio of high-density cholesterol to low-density cholesterol decreased with the increase of dietary calcium and phosphorus content, while other lipid metabolism-related indexes showed no significant difference among the three groups. These results indicate that dietary calcium and phosphorus levels have an effect on lipid metabolism in piglets, especially in terms of lipid accumulation and serum biochemical parameters, it is suggested that the adjustment of calcium and phosphorus in feed may be a potential mechanism affecting lipid metabolism. 3.2 Dietary calcium and phosphorus content regulates intestinal lipid absorption and deposition Lipid metabolism involves the hydrolysis of dietary fats, the absorption of hydrolysates by intestinal cells, and the secretion of coeliac particles and high-density lipoproteins. Special attention has been paid to the intestine, as the first gateway for dietary fats to enter the body, and we have analyzed in depth the jejunum and ileum, the two regions mainly responsible for fatty acid and glycerol absorption. By measuring the lipid content of the jejunum and ileum (Fig. 2 A & B), we found that the levels of triglyceride and cholesterol within the intestinal tissues were proportional to the Ca and P content in the feed, particularly in the HCAP group, the levels of triglyceride in jejunum and ileum were significantly increased ( P < 0.05). Further exploring the regulation of intestinal lipid metabolic pathways, we analyzed the expression of key lipid metabolism genes. The results showed that LCAP significantly inhibited the expression of lipid transport and absorption related genes such as FABP2, CD36, APOB, APOA1, as well as FABP3 and FABP4 in jejunum and ileum (Fig. 2 C & D; P < 0.05). Notably, both high and low calcium-phosphorus diets appeared to suppress FATP4 expression in the jejunum. Meanwhile, low calcium-phosphorus levels also significantly suppressed the expression of fat synthesis-related genes, such as PPARγ and ACC ( P < 0.05), whereas DGAT2 expression tended to decrease but did not reach a significant level. By immunofluorescence technique, we further validated the expression profiles of CD36 and FABP4 proteins in jejunum and ileum (Fig. 2 E), consistent with the results of gene expression analysis. These results together revealed that dietary calcium and phosphorus content had significant effects on intestinal lipid absorption and metabolism. 3.3 Dietary calcium and phosphorus levels regulate intestinal lipid absorption through AMPK/CAMKK2 pathway In order to study the mechanism of calcium and phosphorus regulating lipid absorption, several key parameters of AMPK/CAMKK2 pathway were measured. The expression of AMPKα and its upstream and downstream regulatory genes in the jejunum was significantly suppressed in the low-calcium-phosphate diet group (Fig. 3 A; P < 0.05). The Western blot analysis indicated that the low-calcium-phosphorus diet significantly suppressed the protein levels of phosphorylated AMPKα (P-AMPKα) and sirtuin 1 (SIRT1) in the jejunum (Fig. 3 B; P < 0.05). However, there was no significant difference in the level of phosphorylation of CAMKK2 (P-CAMKK2). The results of immunofluorescence were consistent with the immunoblot analysis, further validating this finding. The results of ileal analysis also supported these findings, showing that the expression of AMPK and its upstream and downstream genes was significantly suppressed under low calcium-phosphorus conditions (Fig. 3 D; P < 0.05). The low calcium-phosphorus diet resulted in a significant decrease in P-AMPKα protein levels (Fig. 3 E&F; P < 0.05), whereas the expression of P-CAMKK2 and SIRT1 proteins showed a decreasing trend but did not reach statistical significance. 3.4 Dietary calcium and phosphorus content regulates liver lipid deposition via the AMPK pathway We first determined the effect on liver TG deposition and showed that low calcium and phosphorus significantly increased TG accumulation in the liver (Fig. 4 a; P < 0.05), but had no significant effect on TC levels (Fig. 4 B). Oil Red O staining further confirmed a significant increase in fat deposition in the liver of piglets in the LCAP group (Fig. 4 C), although liver index did not show a statistical difference (Fig. 4 D). To deeply explore the effects of calcium and phosphorus levels on the regulatory mechanisms of liver lipid metabolism, we analyzed the gene expression of key enzymes and transcriptional regulators of lipid metabolism in the liver. Low calcium and phosphorus levels significantly up-regulated the expression of lipid synthesis-related genes SCD and FASN (Fig. 4 E; P < 0.05), while DGAT1 and SREBP1C showed no significant changes. Meanwhile, the expression of lipolysis-related genes was inhibited and the expression of lipid transport gene CD36 was significantly decreased ( P < 0.05). Immunoblotting results were consistent with gene expression analysis showing that low calcium and phosphorus levels promoted the expression of FASN protein (Fig. 4 F & H), whereas ACC1 and SREBP1-C tended to be elevated (Fig. 4 G&J). There was no significant change in DGAT1 protein expression (Fig. 4 I). 3.5 Dietary calcium and phosphorus content alters liver lipid composition In this study, we report for the first time the effect of dietary calcium and phosphorus levels on liver lipidomic characteristics. Using UPLC-MS non-targeted lipidomic analysis, we detected more than 45 different lipid classes and 3429 different lipid molecules in 18 liver samples from three groups (6 samples per group). By principal component analysis (PCA), we observed significant differences in liver lipid metabolites among the three groups (Fig. 5 A). Further orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that the changes of calcium and phosphorus levels had significantly different effects on liver lipid profiles, this is evident from the predicted separation of principal component 1(Fig. 5 B & C). At the lipid class level, different calcium and phosphorus levels in feeds resulted in changes in the content of specific lipid classes (Fig. 5 D). We found a trend of increased monoradylglycerols (MG) and TG content in the glyceride category in the LCAP group compared with the CON group ( P = 0.09). In the glycerophospholipid category, the levels of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and sphingosine (SPH) were significantly increased, while lysophosphatidylglycerol (LPG) was significantly decreased ( P < 0.05). In the category of nerve sphingolipids, monosyalilated ganglioside M3(GM3) was significantly increased ( P 1 and P -value < 0.05 as screening criteria for significant differences. The results showed that there were 31 differential lipid metabolites in the LCAP group compared with the control group, 22 differential lipid metabolites in the HCAP group compared with the control group, and 8 differential lipid metabolites in the LCAP group compared with the HCAP Group (Fig. 5 E). By analyzing the KEGG pathway for these differential lipid metabolites, we found that calcium and phosphorus levels significantly affected both the Glycerophospholipid pathway and the glyceride pathway (Fig. 5 F). The analysis of lipids showing significant differences between the glyceride pathway (Fig. 5 G) and the Glycerophospholipid pathway (Fig. 5 H) as shown in the heatmap further confirmed the elevation of the glyceride and Glycerophospholipid categories in the LCAP group; This suggests that low levels of calcium and phosphorus disrupt liver lipid metabolism. 3.6 Dietary calcium and phosphorus levels alter gut microbial composition and short-chain fatty acids In order to explore the effects of different dietary calcium and phosphorus levels on the intestinal microbial composition of weaned piglets, we collected colonic content samples from CON, LCAP and HCAP groups, and analyzed them against the bacterial 16S rDNA V3-V4 region using high-throughput sequencing to assess the effects of calcium and phosphorus levels on the structure of intestinal microbial communities. The results of α diversity analysis showed that the CHAO1 index and observed features index in both HCAP and LCAP groups increased, while the goods coverage index decreased, it was suggested that changes in calcium and phosphorus levels in feed might have increased the diversity of gut microbes (Fig. 6 A). The PCoA analysis further confirmed the significant isolation of the flora composition among the three groups (Fig. 6 B). Venn diagram revealed an increase in the total number of OTUs due to changes in calcium and phosphorus levels (Fig. 6 C). Phyla-level microbial relative abundance analysis showed that although the relative abundance of Firmicutes did not change and the ratio of Firmicutes/Bacteroidetes did not differ significantly, the relative abundance of Bacteroidota decreased with increasing levels of calcium and phosphorus (Fig. 6 D). Family-level relative abundance (Fig. 6 E) showed that Prevotellaceae, Streptococcaceae , and Veillonellaceae were negatively correlated with calcium and phosphorus concentrations. Family-level relative abundance analyses revealed that Prevotellaceae , Streptococcaceae , and Veillonellaceae were negatively correlated with calcium and phosphorus concentrations. Compositional analyses at the genus level revealed significantly lower relative abundance of Prevotella_9 in the HCAP group compared to the control group, and significantly lower relative abundance of Alloprevotella compared to the LCAP group ( P < 0.05) (Fig. 6 G). LEfSe analyses identified microbial communities that significantly differed across dietary calcium and phosphorus levels. Micrococcales and g_Lactococcus were dominant in the control group, whereas g_Alloprevotella , g_Streptococcus , and f_Streptococcaceae were dominant in the LCAP group. Tissierellales , Peptostreptococcaceae , Terrisporobacter , and Prevotellaceae_NK3B31_group , on the other hand, were the characteristic flora of the HCAP group (Fig. 6 H). KEGG pathway analyses showed that, compared with the CON and LCAP groups, the HCAP group had a higher predominance in amino acid metabolism, carbohydrate metabolism, lipid metabolism, membrane transport and metabolism pathways were up-regulated in abundance, suggesting that increased calcium and phosphorus levels in the feed contributed to improved lipid metabolism (Fig. 7 A). The results of short-chain fatty acid (SCFA) content showed that low calcium-phosphorus levels significantly reduced the concentrations of isobutyric acid and isovaleric acids, while high calcium-phosphorus levels reduced the concentration of acetic acid (Fig. 7 B), further confirming that calcium-phosphorus levels in feeds have a significant regulatory effect on intestinal metabolites. 3.7 Linkage between the microbiome and lipidomics contributes to the understanding of calcium-phosphorus-regulated lipid metabolism mechanisms. By Spearman's correlation coefficient analysis, we explored the relationship between gut microbial communities and serum glycolipid metabolic indices, which were presented as a heat map (Fig. 7 C). At the genus level, HDL-C showed significant negative correlations with Lachnospiraceae_NK4A136_group , Selenomonas , and Subdoligranulum ( P < 0.0001 to P < 0.001), whereas it showed a positive correlation with Streptococcus ( P < 0.05). In addition, serum TG showed negative correlation with Alloprevotella , Anaerovibrio , Megasphaera ( P < 0.05) and positive correlation with Lachnospiraceae_AC2044_group , Terrisporobacter ( P < 0.05). Through secreting metabolites into the blood, the gut microbiota is involved in the occurrence and progression of diseases[ 12 ]. Spearman correlation analysis revealed a correlation between 21 faecal OTUs and 19 significantly different lipid molecules (Fig. 7 D). Significantly different lipid molecules TG (18:2_13:0_18:2), Cer (d36:0), and PE (18:1_22:1) correlated with a wide range of differentiated bacterial genera. Among them Lachnospiraceae_NK4A136_group , which was previously found to be negatively correlated with serum HDL-C concentration, was significantly positively correlated with the lipid molecule Cer (d46:7). Alloprevotella , which correlated with serum total cholesterol and HDL-C/LDL-C ratio, correlated with TG (18:1_18:1_18:2). 4 Discussion In this study, we systematically investigated for the first time the effects of different calcium and phosphorus levels in the same proportion of feed on lipid metabolism in weaned piglets. It reveals the disorder of lipid metabolism that may result from low calcium-phosphorus levels and elucidates the mechanism of how high-calcium-phosphorus feeds improve lipid metabolism by activating specific signaling pathways. Our results showed that low calcium and phosphorus diets caused significant lipid accumulation in the body and liver of piglets, which was mainly attributed to increased intestinal lipid absorption and abnormal liver lipid accumulation. This finding is consistent with the view of the intestine and liver as the main sites of lipid metabolism and emphasis the importance of regulating the lipid processing capacity of these two organs to maintain lipid homeostasis. Furthermore, our study also showed that by providing a high-calcium phosphate diet, the CAMKK2-AMPKα pathway can be activated, promoting intestinal lipid absorption and expression of transporters, increasing oxidative hydrolysis of renal lipids; At the same time reduce the synthesis of liver lipids, thus effectively improve the lipid metabolic disorder. Combined with the correlation analysis between gut microbial composition and serum lipid metabolism indexes, this study further reveals the important role of gut microbes in the regulation of lipid metabolism. The association of specific microbiota with lipid metabolism-related indicators reinforces the idea that the gut microbiota may be involved in the regulation of lipid metabolism by influencing the secretion patterns of host metabolites. In conclusion, the present study not only highlights the effects of calcium and phosphorus intake on lipid metabolism, but also reveals the potential molecular mechanisms of lipid metabolism regulation, which provides a scientific basis for the development of future nutritional intervention strategies for lipid metabolism disorders. With the further understanding of the mechanism of lipid metabolism, the regulation of calcium and phosphorus intake may become an effective way to improve the disorder of lipid metabolism and prevent related metabolic diseases. Calcium and phosphorus are not only the most abundant minerals in mammals, but are also involved in a variety of key physiological roles, including lipid metabolism and the synthesis and maintenance of bone structure. As a major energy source and active endocrine organ in the body, lipids play a crucial role in the integrity of cell membranes, protection of vital organs, hormone synthesis, and absorption and transport of vitamins, as well as directly affecting livestock and poultry performance and meat quality[ 13 ]. In this study, we found that low calcium and phosphorus diets increased carcass TG and fat percentage, although not significantly, compared with normal calcium and phosphorus levels. This finding echoes previous studies in which calcium supplementation was found to promote muscle fat accumulation[ 14 ], whereas phosphorus supplementation showed the opposite effect[ 4 ]. When calcium and phosphorus were supplemented simultaneously, however, a reduction in muscle lipid accumulation was observed[ 15 ], which is consistent with our experimental results. Notably, adjustment of calcium and phosphorus levels in the feed significantly affected blood glucose concentrations, where high calcium and phosphorus levels significantly reduced blood glucose levels, suggesting that calcium and phosphorus supplementation may play a role in regulating glucose metabolism. Furthermore, we also observed an effect of calcium and phosphorus supplementation on the HDL-C to LDL-C ratio, consistent with Zhang et al[ 16 ]. Although no significant changes were found in other serum lipid metabolic indexes, the decrease of HDL-C/LDL-C ratio may reflect the changes of lipid metabolism. LDL-C plays a key role in cholesterol transport and the synthesis of cell membranes and certain hormones, while HDL-C is responsible for transporting cholesterol in tissues and maintaining the stability of the cardiovascular system. Therefore, changes in the HDL-C/LDL-C ratio may affect cardiovascular health, suggesting a possible role for calcium and phosphorus in maintaining cardiovascular stability[ 17 ]. As the main site of dietary fat absorption, the intestine plays a central role in the overall lipid metabolism. Recent studies have revealed an association between dysregulation of intestinal lipid metabolism and systemic lipid metabolic diseases[ 18 ]. Nevertheless, the specific effects of dietary calcium-phosphorus ratios on intestinal lipid metabolism and their underlying mechanisms are not fully understood. The intestinal lipid accumulation, lipid absorption (CD36, FABP2, FABP3, and FABP4), lipid synthesis (DGAT1, DGAT2, ACC, and PPAR γ) gene and protein expression were detected. Here we reveal a correlation between changes in feed calcium and phosphorus levels and intestinal lipid absorption, pointing to a link between enhanced fatty acid uptake capacity and elevated calcium and phosphorus levels. Furthermore, we observed that low calcium and phosphorus levels promote abnormal accumulation of liver lipids, which results from increased lipid synthesis and inhibition of lipid transport and oxidative hydrolysis of key enzyme activities. In contrast, increased calcium and phosphorus levels mitigated this phenomenon, consistent with previous findings that altering calcium or phosphorus levels alone affects lipid accumulation[ 19 ]. These findings emphasis the important influence of the dietary calcium-phosphorus ratio on the lipid metabolism capacity of the intestine-liver axis. Previous studies on rodents[ 20 ], poultry[ 4 ], fish[ 21 ], and pigs[ 19 ] have all shown that, calcium or phosphorus supplementation alone reduced fat accumulation in the liver and intestines, and we also altered calcium and phosphorus levels in the liver to produce changes consistent with them. The occurrence of this phenomenon may be related to the fact that calcium and phosphorus levels regulate beta oxidation of fatty acids, which is the main pathway of fatty acid degradation. However, the lipid accumulation in the intestine was contrary to previous reports, and the mechanisms need to be further explored because of the paucity of studies on the effects of dietary Ca and P levels on lipid metabolism. AMPK plays a crucial role in the maintenance of cellular energy homeostasis, not only regulating glycolipid metabolism, but also being involved in the modulation of appetite and anorexia signaling[ 7 ]. Although the inhibitory effect of calcium on endogenous lipid production in the liver by activating the AMPK pathway has been reported, its underlying molecular mechanisms are still at the forefront of exploration[ 8 ]. CAMKK2, an upstream kinase of AMPK, plays a critical role in regulating lipid metabolism by phosphorylating AMPK at the Thr172 site. Furthermore, the importance of CaMKK2’s function in regulating lipid metabolism was demonstrated by the ability of its deletion or pharmacological inhibition to reduce ab initio lipogenesis, which shows potential therapeutic value in ameliorating high-fat diet-induced fatty liver, insulin sensitivity problems[9; 10]. As previously reported, elevated levels of dietary calcium and phosphorus increase intracytoplasmic calcium ion concentrations[ 22 ] and serum lipocalin concentrations[23; 24], and lipocalin induces activation of the lipocalin receptor 1 ( AdipoR1 ), which drives CaMKK2 to increase AMPK activity by activating extracellular Ca 2+ efflux[ 25 ]. We further hypothesized that dietary calcium and phosphorus levels might regulate intestinal-liver axis lipid metabolism through the CaMKK2/AMPK signaling pathway. We observed that low calcium-phosphorus levels decreased mRNA and protein levels of intestinal CaMKK2 and AMPKα, accompanied by a decrease in lipid deposition, while high calcium-phosphorus levels reversed this trend. This phenomenon may be related to the upregulation of AMPK activity, which enhances the uptake capacity of long-chain fatty acids (LCFA) in the gut by promoting the expression and membrane translocation of CD36 and FATP4 proteins; This leads to lipid accumulation [ 26 ]. Furthermore, we found that dietary calcium and phosphorus supplementation activated the liver CaMKK2/AMPK signaling pathway to mitigate abnormal accumulation of liver lipids by reducing lipid synthesis and enhancing lipid hydrolytic oxidation processes; This process involves the upregulation of SIRT1, a signaling molecule downstream of AMPK. Our results suggest that dietary calcium and phosphorus levels regulate intestinal-liver axis lipid metabolism through the CaMKK2/AMPK signaling pathway. Given that Ca and P supplementation is known to influence lipid metabolism in skeletal muscle and adipose tissue[1; 14], the present study examined the effects of changes in calcium and phosphorus dietary levels on liver lipid composition, which is important for understanding the role of calcium and phosphorus in overall metabolic regulation. Our findings reveal that low calcium phosphorus intake is associated with increased levels of triglycerides (MG and TG) and certain phospholipids (PIP 2 and SPH) and sphingolipids (GM3 and SM) in the liver; The concentrations of LPG and Ceramide (Cer) were also reduced. This finding implies that dietary calcium and phosphorus levels are negatively correlated with specific lipid classes in the liver. Through the KEGG pathway analysis of differential lipids, we further verified that these lipids are mainly involved in glyceride and Glycerophospholipid metabolic pathways. The increase of glycerides, especially MG and TG, may reflect the accumulation of intermediate products during lipolysis and the enhancement of activity of TG biosynthesis pathway, this is consistent with existing studies of the effects of calcium and phosphorus supplementation alone on lipid metabolism. In addition, the up-regulation of PIP 2 and SPH, as well as the increase in GM3 and SM, may involve changes in cell signaling, cell proliferation and apoptosis, as well as cell membrane composition and function. In particular, changes in SPH and SM implicate a potential role for sphingolipid metabolism in regulating energy homeostasis and AMPK expression. The changes of SPH and SM may affect the energy metabolism of liver by affecting the physical properties of cell membrane and cell signaling pathway[ 27 ]. In particular, SM (d18:1/16:0) was found to enhance ATP production and reduce AMPK expression by activating glycolytic pathways[ 28 ]. This provides new insights into the interaction between lipid metabolism and energy sensing. To establish a potential link between dietary Ca, P, and the microbiome and specific lipid species, we performed a microbiome analysis of colonic contents in the LCAP, HCAP, and CON groups. We found that both the LCAP and HCAP groups increased the alpha diversity of the colonic microbiota compared to the CON group. This may reflect changes in microbial community structure leading to an increase in the diversity of harmful bacteria[ 29 ]. Indeed, a closer examination of the individual bacterial composition of LCAP and HCAP revealed an increased abundance of bacteria belonging to the Enterobacteriaceae , Streptococcaceae , and Clostridiaceae , which are families of bacteria that contain pathogenic bacteria [ 30 ]. The β-diversity analysis further revealed significant differences in microbiome structure among the three groups, consistent with previous findings[ 31 ]. Our examination of diet-related phylum abundance showed that changes in dietary calcium and phosphorus did not alter the abundance of the Firmicutes , but the abundance of the Bacteroidetes declined with increasing Ca and P levels, with the Firmicutes / Bacteroidetes ratio being lowest in the LCAP group. Further analyses of specific ASVs showed that bacteria from the Prevotellaceae and Veillonellaceae families decreased with increasing calcium and phosphorus levels, consistent with their association in obesity pathology[32; 33]. Strikingly, the significant reductions in Alloprevotella and Prevotella in the HCAP group compared with the LCAP group were associated with improvements in liver steatosis and other lipid metabolic markers. Alloprevotella and Prevotella_9 belong to the genus Mycobacterium , which is a group of two in the genus Prevotella . Several reports have linked Prevotella to a range of diseases, including advanced liver fibrosis, cirrhosis, insulin resistance, type 2 diabetes, inflammation and obesity[34; 35]. Furthermore, high abundance of Prevotella copri in a porcine model was associated with elevated concentrations of obesity-related serum metabolites, which were significantly correlated with fat accumulation in pigs[ 36 ]. These results support our observations in this study. Our results provide new insights into the potential role of the gut microbiota in influencing lipid metabolism through calcium and phosphorus levels. The endocrine function of the gut plays a key role in the regulation of lipid metabolism, in particular, short-chain fatty acid produced by the fermentation of gut microbes (SCFA) has a significant effect on lipid biosynthesis through the intestine-liver axis[37; 38]. We observed a decreasing trend in total SCFA levels in colonic contents with increasing dietary calcium and phosphorus levels. This finding echoes previous studies, which have shown that the majority of liver lipogenesis is dependent on microbial SCFA from the colon, which are key components of fatty acid biosynthesis[ 39 ]. Further analyses of specific SCFA concentrations revealed significant reductions in acetic acid concentrations in the HCAP group as well as isobutyric and isovaleric acid concentrations in the LCAP group. Acetic acid as the main energy source of the liver, isobutyric acid and isovalerate play critical roles in maintaining intestinal health, providing energy, having anti-inflammatory and immunomodulatory effects, and promoting the integrity of gut barrier function[ 40 ]. Their decline in LCAP seems to be explained by an increase in pathogenic bacteria. Collectively, our results suggest that dietary calcium and phosphorus levels may provide a feasible path to improve liver steatosis and optimize lipid metabolic indices by influencing SCFA production. It should be noted that final weight, average daily gain and average daily feed intake are not included in this paper, but they have been assessed and will be part of another paper under consideration elsewhere. 5 Conclusion In summary, our results demonstrate that diets low in calcium and phosphorus reduce intestinal lipid absorption and concurrently lead to abnormal accumulation of lipids in the liver and carcass. Conversely, supplementation with calcium and phosphorus can regulate lipid metabolism by activating the CAMKK2/AMPK pathway, promoting the transport of intestinal lipids and the hydrolytic oxidation of liver lipids, and improving the composition of the intestinal microbiota. Overall, we report on the response of intestinal-liver axis lipid metabolism to dietary calcium and phosphorus levels and its regulatory mechanisms, and reveal how these changes affect liver lipid composition and the characteristics of the intestinal microbiome (Fig. 8 ). These findings underscore the importance of precisely managing dietary mineral levels to optimize lipid metabolism and enhance the overall health of animals. Furthermore, given the current lack of approved pharmacological treatments for non-alcoholic fatty liver disease (NAFLD), understanding the mechanisms of action of natural mineral elements provides further insights for the development of drugs to prevent and treat NAFLD 6 Abbreviations ACC1 Acetyl-coa Carboxylase 1 AMPK 5′-Adenosine monophosphate-activated protein kinase AMPKα AMP-activated protein kinase alpha Ca Calcium CAMKK2 Calcium/calmodulin-dependent protein kinase kinase 2 CD36 Cluster of Differentiation 36 Cer Ceramide CPT1A Carnitine palmitoyltransferase 1a DGAT1 Diacylglycerol O-acyltransferase 1 FABP2 Fatty acid binding protein 2 FABP3 Fatty acid binding protein 3 FASN Fatty acid synthase FATP4 Fatty acid transport protein 4 GLU Glucose GM3 Monosyalilated ganglioside M3 HDL-C High-density lipoprotein cholesterol LDL-C Low-density lipoprotein cholesterol LPG Lysophosphatidylglycerol MG Monoradylglycerols NAFLD Nonalcoholic fatty liver disease OTU Operational taxonomic unit P Phosphorus P-AMPKα Phosphorylated AMPKα P-CAMKK2 Phosphorylated CAMKK2 PIP 2 Phosphatidylinositol 4,5-bisphosphate PPARγ Peroxisome proliferator-activated receptor γ PRDM16 PR domain-containing protein 16 SCD Stearoyl-CoA Desaturase SCFA Short chain fatty acid SIRT1 Sirtuin 1 SM Sphingomyelin SPH Sphingosine SREPB1C Sterol regulatory element-binding protein 1c STTD Standardized total tract digestibility TC Total cholesterol TG Triglyceride Declarations Acknowledgements We would like to thank the researchers in our laboratory for their efforts and all the staff at the Baiyun Teaching Experimental Base of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences for their selfless support. Author Contributions Z.M.: Conceptualization, Investigation, Methodology, Data curation, Project administration, Visualization, Writing-original draft. Y.S.: Data curation, Visualization, Writing-original draft. Z.F.: Investigation, Investigation, Data curation. Q.W., X.Y., L.W.: Data curation, Visualization, Writing-original draft. Y.L., Z.J.: Investigation, Methodology, Data curation. H.Y.: Writing-review and editing. Funding This study was financially supported by the National Key Research and Development Program of China (2021YFD1300402), Research Fund of Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture (No. 2022ZD003), the earmarked fund for China Agriculture Research System (CARS-35), and Special Project for Rural Revitalization Strategy in Guangdong Province (2023TS-3-1). Availability of data and materials The data analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All animal procedures were carried out in accordance with the guidelines for the care and use of experimental animals of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences, and approved by the Animal Care Advisory Committee of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Footnotes Hongbo Yi and Ying Li contributed equally to this work. References Grundmann SM, Ress K, Zimmermann L, Horing M, Liebisch G, Most E et al. A high-phosphorus diet moderately alters the lipidome and transcriptome in the skeletal muscle of adult mice. Nutrients. 2023;15(17). doi:10.3390/nu15173734. Mulet-Cabero AI, Wilde PJ. Role of calcium on lipid digestion and serum lipids: a review. Crit Rev Food Sci Nutr. 2023;63(6): 813-26. doi:10.1080/10408398.2021.1954873. 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Supplementary Files Supplementaryinformation1.docx Supplementaryinformation2.docx Cite Share Download PDF Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Journal of Animal Science and Biotechnology → Version 1 posted Editorial decision: Major revision 24 Apr, 2024 Reviewers agreed at journal 02 Apr, 2024 Reviewers invited by journal 17 Mar, 2024 Editor assigned by journal 12 Mar, 2024 First submitted to journal 12 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4069024","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":280660377,"identity":"9ac13f80-d790-4ef5-b052-0cb28ff14955","order_by":0,"name":"zhenyan miao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBACfvbmAwc+GPxj5mdvIFKLZM+xxIczKg6wS/YcIFKLwQ0fY2OeMwf4DWYkEK2Fx0yCt+2OtIHk4403GGpsogk77HZbmYRk2zNjc+m0YguGY2m5DYS08N05vE3CsI052XJ2jpkEY8NhwloYbiSYSSS2MddvuHmGSC0CN1KMDQ6cOcwM9hRRWsCB3FCRxizZA/RLAjF+AUXl4T8GNsCoPLzxxocaGyL8ggQMJBJIUQ7RQqqOUTAKRsEoGBkAALp/RYz/AyF9AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0004-0333-9672","institution":"South China Agricultural University College of Veterinary Medicine","correspondingAuthor":true,"prefix":"","firstName":"zhenyan","middleName":"","lastName":"miao","suffix":""},{"id":280660378,"identity":"466d7610-9e5c-465d-972b-f2f98c7023e3","order_by":1,"name":"Yanjie Sun","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yanjie","middleName":"","lastName":"Sun","suffix":""},{"id":280660379,"identity":"c982ecc3-c38d-4d4b-9d45-a89829584742","order_by":2,"name":"Zhangjian Feng","email":"","orcid":"","institution":"South China Agricultural University College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhangjian","middleName":"","lastName":"Feng","suffix":""},{"id":280660380,"identity":"5ae91c73-b85b-4840-bb15-985dc78e1cb8","order_by":3,"name":"Qiwen Wu","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qiwen","middleName":"","lastName":"Wu","suffix":""},{"id":280660381,"identity":"de94f9df-5385-4730-88ab-da9d7ae0b2f4","order_by":4,"name":"Xuefen Yang","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xuefen","middleName":"","lastName":"Yang","suffix":""},{"id":280660382,"identity":"157b28da-d70c-4817-8350-404490d75814","order_by":5,"name":"Li Wang","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Wang","suffix":""},{"id":280660383,"identity":"f2c588b4-f15a-43e5-94b6-03231e49aee7","order_by":6,"name":"Zongyong Jiang","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zongyong","middleName":"","lastName":"Jiang","suffix":""},{"id":280660384,"identity":"86423f9a-1972-4244-b296-6008919a6b07","order_by":7,"name":"Ying Li","email":"","orcid":"","institution":"South China Agricultural University College of Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Li","suffix":""},{"id":280660385,"identity":"2de995d4-a091-4142-b23a-d64d9d2e8474","order_by":8,"name":"Hongbo Yi","email":"","orcid":"","institution":"Guangdong Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Yi","suffix":""}],"badges":[],"createdAt":"2024-03-11 02:44:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4069024/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4069024/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40104-024-01061-0","type":"published","date":"2024-08-05T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53115412,"identity":"a31c1ed2-4fa7-43d8-a667-4d8d9f4c73a4","added_by":"auto","created_at":"2024-03-20 19:10:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282751,"visible":true,"origin":"","legend":"\u003cp\u003eDietary calcium and phosphorus levels affect carcass and blood lipids. (A) Experimental procedure; (B) Percentage of total fat in carcass; (C) Total triglyceride content in carcass; (D) Serum glucose, total triglyceride, total cholesterol, HDL-C, and LDL-C levels, and the ratio of HDL-C to LDL-C. Data are means and standard errors for six pigs per treatment. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). CON, control group; LCAP, low-calcium-phosphorus group; HCAP, high-calcium-phosphorus group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/e86699391fca7a0457b40508.png"},{"id":53115007,"identity":"93a05348-1cca-4910-ae4e-2b6ef8b8c8ea","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1694196,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of dietary calcium and phosphorus levels on intestinal lipid absorption. (A\u0026amp;B) Total triglyceride and total cholesterol levels in the jejunum ileum. (C\u0026amp;D) Lipid transport (CD36, FABP1, FABP2, FABP3, FABP4, APOA1, APOB) and lipid synthesis (DGAT2, ACC, PPARγ) gene expression abundance in the jejunum and ileum. Data are means and standard errors for six pigs per treatment. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). con, control group; LCAP, low calcium and phosphorus group; HCAP, high calcium and phosphorus group; CD36, Cluster of Differentiation 36; FABP1, fatty acid binding protein 1; FABP2, fatty acid binding protein 2; FABP3, fatty acid binding protein 3; FABP4, fatty acid binding protein 4; APOA1, apolipoprotein A1; APOB, apolipoprotein B; DGAT2. diacylglycerol O-acyltransferase 2; ACC, acetyl CoA carboxylase; PPARγ, peroxisome proliferator-activated receptor gamma.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/9406e02645c006d9d97ec67b.png"},{"id":53115004,"identity":"c1bd6265-3ad5-49cb-9dfc-3629d9f5cb05","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1030216,"visible":true,"origin":"","legend":"\u003cp\u003eDietary calcium and phosphorus levels regulate intestinal lipid metabolism through the CAMKK2/AMPK pathway. (A) Expression abundance of AMPKα and its upstream and downstream genes in the jejunum. (B) Quantification of AMPKα signaling-associated protein bands and protein expression abundance in the jejunum and monitoring with β-actin. (C) Immunofluorescence validation of jejunal AMPKα protein. (D) Expression abundance of AMPKα and its upstream and downstream genes in the ileum. (E) Quantification of AMPKα signaling-associated protein bands and protein expression abundance in ileum and monitoring with β-actin. (F) Immunofluorescence validation of ileal AMPKα protein. Data are means and standard errors for six pigs per treatment. Different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). CON, control group; LCAP, low calcium-phosphorus group; HCAP, high calcium-phosphorus group; AMPKα, AMP-activated protein kinase alpha; CAMKK2, calcium/calmodulin-dependent protein kinase kinase 2; SIRT1, sirtuin 1.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/8fb85e3f500aa6ce5cc38f86.png"},{"id":53115006,"identity":"86d115d2-4b29-48da-9dd5-3e809dac9f90","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":699143,"visible":true,"origin":"","legend":"\u003cp\u003eDietary calcium and phosphorus content regulates liver lipid metabolism through the CAMKK2/AMPK pathway. (A) Liver total cholesterol content. (B) Liver total triglyceride content. (C) Oil red O staining showing liver lipid accumulation. (D) Liver index. (E) Liver lipid synthesis (SCD, FASN, DGAT1, SREPB1C), lipid hydrolysis and oxidation (ATGC, PPARα, CPT1A, PRDM16), and lipid transport (CD36, FABP4) gene expression abundance. (F) Liver lipid synthesis-related protein bands. (G-J) Protein expression associated with liver lipid synthesis. (K) Liver CAMKK2/AMPK pathway gene expression abundance. (L-N) Protein levels of p-AMPK and p-CAMKK2 were examined by protein blot analysis and monitored with AMPK and CAMKK2, respectively. Data are means and standard errors of six pigs per treatment; different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05); CON, control; LCAP, low calcium-phosphorus group; HCAP, high calcium-phosphorus group; SCD, stearoyl-CoA desaturase; FASN, fatty acid synthase; DGAT1, diacylglycerol O-acyltransferase 1; SREBP1C, sterol regulatory element-binding protein 1C; ATGC, Acyl-CoA thioesterase 4; PPARα, peroxisome proliferator-activated receptor alpha; CPT1A, carnitine palmitoyltransferase 1A; PRDM16, PR domain containing 16; CD36, cluster of differentiation 36; FABP4, fatty acid-binding protein 4.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/d8227155d8ac168a11bf4aef.png"},{"id":53115011,"identity":"f38f987f-4565-4f27-ac09-0d74afd06e08","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":492127,"visible":true,"origin":"","legend":"\u003cp\u003eLiver untargeted lipidomic analysis. (A) PCA analysis (B-C) OPLS-DA analysis of HCAP vs CON group and LCAP vs CON group. (D) Content of glycerolipids, glycerophospholipids, sphingolipids, and fatty acyl groups in CON, LCAP, and HCAP groups; n = 6; error lines represent SEM.(E) Volcano plots of two-by-two comparisons between the three groups. (F) Metabolic pathway analysis of differential lipids using MetaboAnalyst. (G) Heat map of relative abundance of glycerol ester differential lipids in the three groups. (H) Heat map of relative abundance of glycerophospholipid differential lipids in the three groups. Data are means and standard errors for six pigs per treatment; different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/64ebaffed567acd2d8640a9d.png"},{"id":53115010,"identity":"8d2681c6-6d05-49f1-85ec-046404dad1eb","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":585601,"visible":true,"origin":"","legend":"\u003cp\u003eDietary calcium and phosphorus levels influence the structure of the gut microbiota. (A) α-diversity of the gut flora. (B) β-diversity: principal coordinate analysis (PCoA) based on OTU abundance per weaned piglet. (C) Venn diagram based on OTU levels. (D) Relative abundance of gut microbiota at the phylum level and (E) family level. (F) Heat map based on genus level. (G) Changes in flora associated with lipid metabolism: level of \u003cem\u003eFirmicutes/Bacteroidetes\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e abundance, \u003cem\u003ePrevotella_9\u003c/em\u003e abundance, \u003cem\u003eLactobacillus\u003c/em\u003e abundance. (H) Linear discriminant analysis of effect size (LEfSe) from gate level to genus level (LDA \u0026gt; 3.0). Data are expressed as mean ± SEM and compared with one-way analysis of variance (ANOVA) by Tukey's multiple comparisons post-test.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/fc36460921d2fbbc93c61d10.png"},{"id":53115012,"identity":"e7942da5-fb91-4b79-9d8d-a375703fd081","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":518076,"visible":true,"origin":"","legend":"\u003cp\u003eLink between microbiome and lipidomics at different dietary calcium and phosphorus levels. (A) Heat map showing the gut microbiota predicting changes in KEGG pathways at different calcium and phosphorus levels. (B) SCFA (\u003cem\u003en\u003c/em\u003e = 6 per group) including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid pentanoic acid, hexanoic acid, and total short-chain fatty acids were determined in the colonic contents of weaned piglets by gas chromatography; different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). (C) Heatmap showing Spearman correlation analysis between gut microbiota and serum lipid metabolism indices. Red represents positive correlations and blue represents negative correlations. Significant correlations are marked with *(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), **(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) and ***(\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001). (D) Relationship between discriminatory colonic contents microbial OTUs and significantly different lipid molecules at different calcium and phosphorus levels. The size of the dot for each genus shows the average relative abundance. Dots represent faecal microbiomes and square dots represent lipid molecules. Transparency of the line indicates the negative logarithm of the correlation \u003cem\u003eP\u003c/em\u003e-value (Spearman's) (bottomed out at 10), the green line indicates a negative correlation, the orange line indicates a positive correlation, and the width of the line indicates a large correlation (Spearman's).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/1bfc5b571fe35cfb25c6532b.png"},{"id":53115009,"identity":"b993966c-eeff-4e8d-a3b8-2143f294609c","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":530902,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of dietary calcium and phosphorus levels regulating lipid metabolism in weaned piglets. Calcium and phosphorus in the diet regulate intestinal lipid absorption and hepatic lipid metabolism through the CAMKK2/AMPK axis and alter the gut microbiota to influence lipid metabolism.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/361da158b3e6c8e171b09653.png"},{"id":62298580,"identity":"f8fe8d32-1ee6-470e-a55a-061b33bd3b67","added_by":"auto","created_at":"2024-08-12 16:14:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6425784,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/00b7273d-0780-4934-aa5e-7f87b2a8279a.pdf"},{"id":53115008,"identity":"aaa78add-3f9e-4c93-ac6a-de611fb8f10a","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":40820,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/c1617dd802a6f8d4036fe5e8.docx"},{"id":53115014,"identity":"612727c3-3b94-46d3-9a37-5f213092c1e4","added_by":"auto","created_at":"2024-03-20 19:02:24","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1563891,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4069024/v1/d2952210b385d5298ee2022b.docx"}],"financialInterests":"","formattedTitle":"CAMKK2-AMPK axis endows dietary calcium and phosphorus levels with regulatory effects on lipid metabolism in weaned piglets","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCalcium and phosphorus, fundamental to the mineral nutrient spectrum, play an indispensable role in bone development and act as critical secondary messengers in cellular signaling. The significance of these minerals transcends the mere provision of skeletal integrity, extending to a broad spectrum of physiological processes, including muscle contraction, the facilitation of neurotransmitter dissemination, hormone secretion, and the regulation of body weight equilibrium. These roles underscore the indispensable contribution of calcium and phosphorus to both developmental and homeostatic mechanisms within the biological system[1; 2]. In the field of lipid metabolism, calcium and phosphorus are essential for the maintenance of a healthy state. Calcium augments energy utilization and weight regulation through the facilitation of lipolysis, amplification of insulin sensitivity, meticulous regulation of fatty acid oxidation, and nuanced modulation of cholesterol levels. On the other hand, phosphorus occupies a central position in energy metabolism. The role of phosphorus in ATP production is well-established, significantly impacting lipid synthesis and catabolism, lipoprotein metabolism, and, through the regulation of hormones like insulin, subsequently influencing the overarching lipid equilibrium. Calcium supplementation has been shown to mitigate organ fat accumulation induced by a high-fat diet and to enhance intramuscular fat storage in livestock, indicating its potential in improving meat quality and animal health[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Conversely, whilst phosphorus supplementation was able to reduce overall lipid content, it also reduced intramuscular fat content, demonstrating its subtle effects on lipid distribution within muscle tissue[1; 4]. Previous studies have focused on the dual regulation of calcium and phosphorus on muscle performance, leaving a gap in understanding the effects of dietary calcium and phosphorus changes on overall lipid metabolism and the mechanisms involved. This knowledge gap underscores the need for a more comprehensive exploration of how these minerals interact in complex networks of lipid metabolism that may provide new nutritional strategies for animal health and production performance.\u003c/p\u003e \u003cp\u003eIn the animal body, lipids play a multifaceted and critical role, not only as a major energy reservoir, but also influencing livestock production and meat quality. However, excessive accumulation of fat not only reduces lean meat percentage and meat quality, but also affects feed conversion efficiency and may even lead to serious health problems such as fatty liver[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In animals, fats are primarily synthesized through the esterification of fatty acids with glycerol, serving as the main source of energy reserves in the body. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Lipids not only serve as the energy base necessary for the maintenance of daily physiological activities, but also play key roles in a variety of biological processes, including thermoregulation, construction and maintenance of cell membrane structure and function, regulation of insulin sensitivity, protection of vascular health, and participation in immune responses[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition, adipose tissue is an organ with important endocrine functions. It plays a key role in the regulation of metabolic health and energy balance through the secretion of a variety of bioactive substances, such as lipocalins, resistin and leptin. Therefore, understanding and controlling fat accumulation and its metabolic processes are important for improving meat quality, enhancing animal health, and improving feed efficiency.\u003c/p\u003e \u003cp\u003eAs a central regulator of intracellular energy metabolism, 5\u0026prime;-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is essential for maintaining the stable availability of glucose, glycogen and fatty acids. In addition, AMPK plays a key role in signaling pathways that sense intracellular lysosomal and nuclear DNA damage[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Studies have shown that AMPK has an important role in the regulation of lipid and glucose metabolism in the heart, hypothalamus, adipose tissue, muscle, and liver. Calcium has a marked inhibitory effect on endogenous lipid production in the liver through activation of the AMPK pathway, but the deeper molecular mechanisms have not been elucidated[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. As an upstream signal of AMPK, calcium/calmodulin-dependent kinase kinase 2 (CAMKK2) can enhance its regulatory effect on lipid metabolism by activating AMPK, and conversely AMPK activation can influence Ca\u0026sup2;⁺ signaling and modulate CaMKK2 activity. The function of CaMKK2, especially in regulating lipid metabolism has been demonstrated in several studies. Both genetic deletion and pharmacological inhibition of CaMKK2 significantly reduces de novo fatty acid synthesis and may bring about amelioration of high-fat diet-induced fatty liver, reduced insulin sensitivity, and prostate cancer cell proliferation[9; 10].\u003c/p\u003e \u003cp\u003eAlthough calcium and phosphorus, as key mineral nutrients, play important roles in maintaining the body's lipid metabolism homeostasis, the understanding of how different dietary calcium and phosphorus intakes specifically affect lipid metabolism and their molecular mechanisms is incomplete. In view of this, the aim of this study was to investigate the effects of different calcium and phosphorus levels (normal, low and high) on lipid metabolism and the mechanisms behind them using a weaned piglet model. In particular, this study focused on the interaction between intestine and liver during lipid absorption and processing, revealing how increased calcium and phosphorus levels ameliorate disordered lipid metabolism by activating the AMPK/CAMKK2 pathway in the intestine-liver axis. Further, this study also examined the correlation between gut microbiota and liver lipid composition, exploring how the intestine-liver axis serves as a key mechanism by which dietary calcium and phosphorus regulate lipid metabolism. This comprehensive study not only deepens our understanding of the role of calcium and phosphorus in the regulation of lipid metabolism, but also provides new insights into how to optimize lipid metabolism by modulating calcium and phosphorus intake in the diet.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethics Statement\u003c/h2\u003e \u003cp\u003e All animal procedures were carried out in accordance with the guidelines for the care and use of experimental animals of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences, and approved by the Animal Care Advisory Committee of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals and experimental protocol\u003c/h2\u003e \u003cp\u003eBefore the study began, all the components of Ca and P used to produce the experimental diet were measured in the laboratory, the standardized total tract digestible (STTD) Ca and STTD P values of feeds were calculated based on the STTD Ca and STTD P data of feedstuffs for further formulation of feed formulations. The basal ration was a corn-soybean meal type ration, the ratio of dietary STTD Ca to STTDP was fixed at 1.2, STTD Ca varied with STTD P, and other nutrient levels were formulated with reference to NRC(2012), and the composition of the diets is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. In order to eliminate the effect of phytase, phytase was not added in the experiment.\u003c/p\u003e \u003cp\u003eSeventy-two weaned piglets (\u003cem\u003eDuroc\u003c/em\u003e \u0026times; \u003cem\u003eLandrace\u003c/em\u003e \u0026times; \u003cem\u003eYorkshire\u003c/em\u003e, 25 days of age) with an initial body weight (BW) of 7.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 kg were randomly assigned to three treatment groups, with six replicate pens of four weaned piglets per treatment. Control (CON) pigs were fed a diet with an STTD Ca of 0.504 and an STTD P of 0.42. Pigs in the low calcium-phosphorus group (LCAP) were fed a diet with an STTD Ca of 0.216 and an STTD P of 0.18. Pigs in the high calcium-phosphorus group (HCAP) were fed a diet with an STTD Ca of 0.696 and an STTD P of 0.58. Animals are fed in separate enclosures (of the same size). All pigs are fed three times a day and are given clean water free of charge. The experiment lasted six weeks. Food intake was recorded daily and initial and final weight was measured at the beginning and end of the experiment, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sample collection\u003c/h2\u003e \u003cp\u003ePrior to slaughter, blood samples were collected and centrifuged for 12 min (3000rpm/min, 4\u0026deg;C) to obtain serum, which was stored at -4\u0026deg;C for further determination. The intestinal, muscle, chyme and liver tissues were rapidly frozen in liquid nitrogen immediately after slaughter and stored at -80\u0026deg;C for further study. A portion of liver, intestine and muscle tissues were fixed in 4% paraformaldehyde for histological observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Carcass chemical analysis\u003c/h2\u003e \u003cp\u003eFirst, the whole carcass (n\u0026thinsp;=\u0026thinsp;6) including the bones of the three groups of piglets were frozen and processed into bone paste by using different sizes of grinders. The chopped samples were thoroughly homogenized in a blender. Samples (500 g) were then collected, vacuum packed and stored at -20\u0026deg;C until further analysis. The total fat content of carcass was determined by Soxhlet extraction method. Briefly, three replicates of each sample were taken and about 30 g of carcass samples were ground into minced meat, dried in a vacuum freeze dryer and then ground into powder. One g of dried meat sample (accurate to 0.0001 g) was wrapped in filter paper into a cylindrical filter cup and extracted with n-hexane at 140\u0026deg;C for 50 min in a Soxhlet extraction unit (SE-A6, alva, Jinan, China). After air-drying for 10 min and baking at 102\u0026deg;C for 30 min, the extracted oil-containing aluminium cups were accurately weighed (to the nearest 0.0001 g) when the aluminium cups were cooled to room temperature. Total carcass fat (%) = (weight of oil-containing aluminium cups after extraction - weight of empty aluminium cups)/weight of air-dried meat samples \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biochemical analysis\u003c/h2\u003e \u003cp\u003eSerum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) concentrations, and glucose (GLU) concentrations were determined using an automated biochemical analyser (ICUBIO, iMagic-M7, Shenzhen, China).\u003c/p\u003e \u003cp\u003eIntestinal samples, liver samples, muscle samples and carcass samples were homogenised in saline solution (1:9) and sediment was removed by centrifugation (3000 rpm, 10 min) to obtain 10% tissue homogenate. The TC and TG levels in the intestines, liver, muscles, and torso were all measured using colorimetric assay kits(Nanjing Jiancheng Biotechnology Company, Nanjing, China) according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Histological analysis\u003c/h2\u003e \u003cp\u003eThe liver tissues of piglets fixed in 4% paraformaldehyde were frozen and embedded. The cryosections were cut into 10 \u0026micro;m thickness and stained with Oil red O. The sections were washed with 85% propylene glycol and then with distilled water before being stained with hematoxylin. The staining process was repeated after each wash. The presence of lipid droplets was indicated by a red stain. Sections were examined under a microscope (Nikon, Tokyo, Japan), photographed and recorded using Image-Pro Plus 6.0 software for subsequent comparison.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Real-time quantitative PCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA from tissues was extracted with TRIzol reagent (Takara Bio, Shiga, Japan). 1\u0026micro;g of RNA was then reverse transcribed to cDNA using the PrimeScript RT kit with cDNA Eraser (Takara Bio) according to the kit instructions. 1\u0026micro;g of RNA was extracted from the tissue or cells with the fluorescence quantification kit SYBR\u0026reg; Premix Ex Taq (TaKaRa Bio) was used to perform real-time PCR on the CFX Connect Detection System (Bio-Rad, Hercules, CA, USA).The 20\u0026micro;L of reaction system showed as below, SYBR Premix Ex Taq (10\u0026micro;L), upstream primer (0.4\u0026micro;L), upstream primer (0.4\u0026micro;L), downstream primer (0.4\u0026micro;L), ROX II dye (0.4\u0026micro;L), cDNA samples (2\u0026micro;L), and ultrapure water (6.8\u0026micro;L).\u003c/p\u003e \u003cp\u003eThe real-time quantitative PCR reaction conditions were: pre-denaturation at 95\u0026deg;C for 5 min, denaturation at 95\u0026deg;C for 5 s, annealing at 60\u0026deg;C for 34 s, extension at 95\u0026deg;C for 15 s, and the number of cycles of amplification was 40. The results were calculated by the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, and all the primer sequences required for qPCR in this study were shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eThe samples were separated by SDS-PAGE and the protein was transferred to PVDF membrane. The following antibodies were used in western blotting, such as β-actin (Affinity, T0022, 1:3000), PPARγ (Abcam, 209350, 1:1000), SIRT1 (Cell Signaling Technology, 9475S, 1:1000), AMPKα (Cell Signaling Technology, #5831, 1:1000), phospho-AMPKα (Thr172) (Cell Signaling Technology, #2535, 1:1000), CAMKK2 (Proteintech, 111549-1-AP, 1:1000), phospho-CaMKK2 (Ser511) (Cell Signaling Technology, #12818, 1:1000), DGAT1(Abcam,ab54037, 1:1000),ACC1(Cell Signaling Technology, #4190, 1:1000), SREPB1C(Abcam, ab28481, 1:1000), and FASN (Cell Signaling Technology, 3180S, 1:1000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eFixed intestinal tissues were embedded in paraffin and cut into 4\u0026micro;m sections, then dewaxed, rehydrated, and treated in microwave oven with EDTA-containing antigen retrieval buffer (PH 8.0). Afterwards, sections were blocked with 5% fetal bovine serum (Bioss, Beijing, China) for 1 h at room temperature and then incubated overnight (1:500 dilution) at 4\u0026deg;C with rabbit anti-AMPKα, CD36, FABP4 (Service Bio, Wuhan, China). After rinsing with PBS, they were incubated with Alexa Fluor 555(BBI, Shanghai, China)-conjugated goat anti-rabbit secondary antibody for 30 min at room temperature in the dark. In addition, images were obtained under a microscope with magnification of 200 (Nikon, Tokyo, Japan) and positive results were quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Non-targeted lipidomics of liver\u003c/h2\u003e \u003cp\u003eThe total lipids were extracted from the livers of three groups of piglets. After thawing, liver tissue (20 Mg) was homogenized in 1 ml mixture (methanol, MTBE, and internal standard mixture). After that, ultrasound was performed at 4\u0026deg;C for 20 minutes and then left at room temperature for 30 minutes. The solution was centrifuged at 10\u0026deg;C at 14000 g for 15 min to obtain an upper organic solvent layer and dried under nitrogen. Samples were analyzed using a high-performance liquid chromatography system (UHPLC Nexera Shimadzu LC-30A) and LC separation was performed on a Waters ACQUITY PREMIERCSH C18 column (1.7\u0026micro;m, 2.1 mm \u0026times; 100 mm). The lipid extract was redissolved in 200 mL of 90% isopropanol/acetonitrile, centrifuged at 14000 g for 15 min, and finally injected with 3 mL of the sample. Solvent A was acetonitrile-water (6:4, v/V) containing 0.1% formic acid and 0.1 mM ammonium formate, and solvent B was acetonitrile-isopropanol (1:9, v/V) containing 0.1% formic acid and 0.1 mM ammonium formate. The initial flow rate was 300\u0026micro;L/min with 40% Solvent B. Hold for 3.5 min, then linearly increase to 75% of solvent B within 9.5 min, then linearly increase to 99% of solvent B within 6 min, then equilibrate in 40% of solvent B for 5 min. The mass spectra were obtained by Q-exactive Plus in both positive and negative modes. All measured ESI parameters were optimized and preset as follows: source temperature, 300\u0026deg;C; capillary temperature, 350\u0026deg;C, ion spray voltage set to 3000 V, and S-Lens RF level set to 50%, respectively, the scanning range of the instrument is set to M/z 200\u0026ndash;1800. Lipid search was used for peak recognition, peak extraction, and lipid identification (secondary identification). Fatty acid composition expressed as a percentage of total fatty acids.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 16S rRNA sequencing and processing\u003c/h2\u003e \u003cp\u003eTotal genomic DNA of the gut microbiota was extracted from colon contents samples using a DNA isolation kit (Omega Bio-Tek, Norcross, GA, USA). DNA concentration was determined using a Nanodrop instrument (Thermo Fisher Scientific). DNA integrity was assessed using 2% agar gel electrophoresis. Universal forward primer (5'-CCTAYGGGGRBGCASCAG-3') and reverse primer (5'-GGACTACNNGGGGTATCTAAT \u0026minus;\u0026thinsp;3') were used to amplify the V3-V4 region of the bacterial 16S rRNA gene. Sequencing was performed on the Illumina MiSeq/NovaSeq platform. The DADA2 module in QIIME2 (Version QIIME2-202202) software was used for noise reduction. Microbial composition diversity was analysed using QIIME and R software. PICRUSt2 software was used for KEGG pathway analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Short-chain fatty acid analysis\u003c/h2\u003e \u003cp\u003eShort-chain fatty acids in colon contents were determined using gas chromatography. To prepare the sample, 50 mg of colon contents were mixed with 250 \u0026micro;L of ultrapure water for 5 minutes. The suspension was then centrifuged at 5000 rpm for 30 minutes. Next, 1 mL of supernatant was transferred to a 2 mL PE tube and mixed with 200\u0026micro;L of 42 mmol/L crotonic acid and 200\u0026micro;L of 10% metaphosphoric acid solution. The PE tubes were refrigerated overnight at 4\u0026deg;C and then centrifuged at 10,000 rpm for 10 minutes at 4\u0026deg;C. The resulting supernatant was mixed with an equal amount of ether and extracted for 5 minutes. The ether layer was aspirated using a disposable syringe, filtered through a 0.22\u0026micro;m organic membrane, and then injected into a brown vial for injection. Short-chain fatty acids (acetic, propionic, butyric, valeric, isobutyric and isovaleric acids, with crotonic acid as an internal standard) were measured using a gas chromatograph and a mass spectrometry detector (7890A and 5975C Inert XL EI/CI Mass Detectors, Agilent Technologies, Santa Clara, CA, USA).Detection of short-chain fatty acids was performed according to a previous GC procedure[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Data analysis\u003c/h2\u003e \u003cp\u003eThe results were statistically analyzed using SPSS 21.0 statistical analysis software, and the data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical treatment was performed using one-way analysis of variance (ANOVA) followed by LSD post-test. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Effect of calcium and phosphorus content on lipid homeostasis\u003c/h2\u003e\n\u003cp\u003eIn a six-week dietary intervention study (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), we systematically evaluated the effects of dietary calcium and phosphorus on lipid homeostasis in piglets. We first measured the percentage of crude fat in the carcass and the level of total triglyceride to assess the direct effect of dietary calcium and phosphorus on lipid accumulation. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the percentage of crude fat in carcasses showed a decreasing trend as the calcium and phosphorus content of the feed increased. In addition, the measurements of total triglyceride levels in carcasses (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) were consistent with the observations of crude fat percentage, suggesting that lipid accumulation was regulated by feed calcium and phosphorus content. Further analysis of serum biochemical parameters of piglets (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD) revealed that in piglets fed with high calcium and phosphorus diets, the serum glucose concentration was significantly lower than that of CON group and LCAP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the ratio of high-density cholesterol to low-density cholesterol decreased with the increase of dietary calcium and phosphorus content, while other lipid metabolism-related indexes showed no significant difference among the three groups. These results indicate that dietary calcium and phosphorus levels have an effect on lipid metabolism in piglets, especially in terms of lipid accumulation and serum biochemical parameters, it is suggested that the adjustment of calcium and phosphorus in feed may be a potential mechanism affecting lipid metabolism.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Dietary calcium and phosphorus content regulates intestinal lipid absorption and deposition\u003c/h2\u003e\n\u003cp\u003eLipid metabolism involves the hydrolysis of dietary fats, the absorption of hydrolysates by intestinal cells, and the secretion of coeliac particles and high-density lipoproteins. Special attention has been paid to the intestine, as the first gateway for dietary fats to enter the body, and we have analyzed in depth the jejunum and ileum, the two regions mainly responsible for fatty acid and glycerol absorption.\u003c/p\u003e\n\u003cp\u003eBy measuring the lipid content of the jejunum and ileum (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; B), we found that the levels of triglyceride and cholesterol within the intestinal tissues were proportional to the Ca and P content in the feed, particularly in the HCAP group, the levels of triglyceride in jejunum and ileum were significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further exploring the regulation of intestinal lipid metabolic pathways, we analyzed the expression of key lipid metabolism genes. The results showed that LCAP significantly inhibited the expression of lipid transport and absorption related genes such as FABP2, CD36, APOB, APOA1, as well as FABP3 and FABP4 in jejunum and ileum (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC \u0026amp; D; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, both high and low calcium-phosphorus diets appeared to suppress FATP4 expression in the jejunum. Meanwhile, low calcium-phosphorus levels also significantly suppressed the expression of fat synthesis-related genes, such as PPAR\u0026gamma; and ACC (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas DGAT2 expression tended to decrease but did not reach a significant level. By immunofluorescence technique, we further validated the expression profiles of CD36 and FABP4 proteins in jejunum and ileum (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE), consistent with the results of gene expression analysis. These results together revealed that dietary calcium and phosphorus content had significant effects on intestinal lipid absorption and metabolism.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Dietary calcium and phosphorus levels regulate intestinal lipid absorption through AMPK/CAMKK2 pathway\u003c/h2\u003e\n\u003cp\u003eIn order to study the mechanism of calcium and phosphorus regulating lipid absorption, several key parameters of AMPK/CAMKK2 pathway were measured. The expression of AMPK\u0026alpha; and its upstream and downstream regulatory genes in the jejunum was significantly suppressed in the low-calcium-phosphate diet group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The Western blot analysis indicated that the low-calcium-phosphorus diet significantly suppressed the protein levels of phosphorylated AMPK\u0026alpha; (P-AMPK\u0026alpha;) and sirtuin 1 (SIRT1) in the jejunum (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, there was no significant difference in the level of phosphorylation of CAMKK2 (P-CAMKK2). The results of immunofluorescence were consistent with the immunoblot analysis, further validating this finding. The results of ileal analysis also supported these findings, showing that the expression of AMPK and its upstream and downstream genes was significantly suppressed under low calcium-phosphorus conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The low calcium-phosphorus diet resulted in a significant decrease in P-AMPK\u0026alpha; protein levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026amp;F; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the expression of P-CAMKK2 and SIRT1 proteins showed a decreasing trend but did not reach statistical significance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Dietary calcium and phosphorus content regulates liver lipid deposition via the AMPK pathway\u003c/h2\u003e\n\u003cp\u003eWe first determined the effect on liver TG deposition and showed that low calcium and phosphorus significantly increased TG accumulation in the liver (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but had no significant effect on TC levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Oil Red O staining further confirmed a significant increase in fat deposition in the liver of piglets in the LCAP group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC), although liver index did not show a statistical difference (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). To deeply explore the effects of calcium and phosphorus levels on the regulatory mechanisms of liver lipid metabolism, we analyzed the gene expression of key enzymes and transcriptional regulators of lipid metabolism in the liver. Low calcium and phosphorus levels significantly up-regulated the expression of lipid synthesis-related genes SCD and FASN (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while DGAT1 and SREBP1C showed no significant changes. Meanwhile, the expression of lipolysis-related genes was inhibited and the expression of lipid transport gene CD36 was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Immunoblotting results were consistent with gene expression analysis showing that low calcium and phosphorus levels promoted the expression of FASN protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF \u0026amp; H), whereas ACC1 and SREBP1-C tended to be elevated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG\u0026amp;J). There was no significant change in DGAT1 protein expression (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Dietary calcium and phosphorus content alters liver lipid composition\u003c/h2\u003e\n\u003cp\u003eIn this study, we report for the first time the effect of dietary calcium and phosphorus levels on liver lipidomic characteristics. Using UPLC-MS non-targeted lipidomic analysis, we detected more than 45 different lipid classes and 3429 different lipid molecules in 18 liver samples from three groups (6 samples per group). By principal component analysis (PCA), we observed significant differences in liver lipid metabolites among the three groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eFurther orthogonal partial least squares discriminant analysis (OPLS-DA) revealed that the changes of calcium and phosphorus levels had significantly different effects on liver lipid profiles, this is evident from the predicted separation of principal component 1(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB \u0026amp; C). At the lipid class level, different calcium and phosphorus levels in feeds resulted in changes in the content of specific lipid classes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). We found a trend of increased monoradylglycerols (MG) and TG content in the glyceride category in the LCAP group compared with the CON group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09). In the glycerophospholipid category, the levels of phosphatidylinositol 4,5-bisphosphate (PIP\u003csub\u003e2\u003c/sub\u003e) and sphingosine (SPH) were significantly increased, while lysophosphatidylglycerol (LPG) was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the category of nerve sphingolipids, monosyalilated ganglioside M3(GM3) was significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the content of SM also tended to increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.076). We further analyzed differences in lipid metabolites among the three groups using OPLS-DA VIP\u0026thinsp;\u0026gt;\u0026thinsp;1 and \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as screening criteria for significant differences. The results showed that there were 31 differential lipid metabolites in the LCAP group compared with the control group, 22 differential lipid metabolites in the HCAP group compared with the control group, and 8 differential lipid metabolites in the LCAP group compared with the HCAP Group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). By analyzing the KEGG pathway for these differential lipid metabolites, we found that calcium and phosphorus levels significantly affected both the Glycerophospholipid pathway and the glyceride pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). The analysis of lipids showing significant differences between the glyceride pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG) and the Glycerophospholipid pathway (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH) as shown in the heatmap further confirmed the elevation of the glyceride and Glycerophospholipid categories in the LCAP group; This suggests that low levels of calcium and phosphorus disrupt liver lipid metabolism.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 Dietary calcium and phosphorus levels alter gut microbial composition and short-chain fatty acids\u003c/h2\u003e\n\u003cp\u003eIn order to explore the effects of different dietary calcium and phosphorus levels on the intestinal microbial composition of weaned piglets, we collected colonic content samples from CON, LCAP and HCAP groups, and analyzed them against the bacterial 16S rDNA V3-V4 region using high-throughput sequencing to assess the effects of calcium and phosphorus levels on the structure of intestinal microbial communities. The results of \u0026alpha; diversity analysis showed that the CHAO1 index and observed features index in both HCAP and LCAP groups increased, while the goods coverage index decreased, it was suggested that changes in calcium and phosphorus levels in feed might have increased the diversity of gut microbes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eThe PCoA analysis further confirmed the significant isolation of the flora composition among the three groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Venn diagram revealed an increase in the total number of OTUs due to changes in calcium and phosphorus levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Phyla-level microbial relative abundance analysis showed that although the relative abundance of Firmicutes did not change and the ratio of \u003cem\u003eFirmicutes/Bacteroidetes\u003c/em\u003e did not differ significantly, the relative abundance of \u003cem\u003eBacteroidota\u003c/em\u003e decreased with increasing levels of calcium and phosphorus (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Family-level relative abundance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE) showed that \u003cem\u003ePrevotellaceae, Streptococcaceae\u003c/em\u003e, and \u003cem\u003eVeillonellaceae\u003c/em\u003e were negatively correlated with calcium and phosphorus concentrations. Family-level relative abundance analyses revealed that \u003cem\u003ePrevotellaceae\u003c/em\u003e, \u003cem\u003eStreptococcaceae\u003c/em\u003e, and \u003cem\u003eVeillonellaceae\u003c/em\u003e were negatively correlated with calcium and phosphorus concentrations. Compositional analyses at the genus level revealed significantly lower relative abundance of \u003cem\u003ePrevotella_9\u003c/em\u003e in the HCAP group compared to the control group, and significantly lower relative abundance of \u003cem\u003eAlloprevotella\u003c/em\u003e compared to the LCAP group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). LEfSe analyses identified microbial communities that significantly differed across dietary calcium and phosphorus levels. \u003cem\u003eMicrococcales\u003c/em\u003e and \u003cem\u003eg_Lactococcus\u003c/em\u003e were dominant in the control group, whereas \u003cem\u003eg_Alloprevotella\u003c/em\u003e, \u003cem\u003eg_Streptococcus\u003c/em\u003e, and \u003cem\u003ef_Streptococcaceae\u003c/em\u003e were dominant in the LCAP group. \u003cem\u003eTissierellales\u003c/em\u003e, \u003cem\u003ePeptostreptococcaceae\u003c/em\u003e, \u003cem\u003eTerrisporobacter\u003c/em\u003e, and \u003cem\u003ePrevotellaceae_NK3B31_group\u003c/em\u003e, on the other hand, were the characteristic flora of the HCAP group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH). KEGG pathway analyses showed that, compared with the CON and LCAP groups, the HCAP group had a higher predominance in amino acid metabolism, carbohydrate metabolism, lipid metabolism, membrane transport and metabolism pathways were up-regulated in abundance, suggesting that increased calcium and phosphorus levels in the feed contributed to improved lipid metabolism (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). The results of short-chain fatty acid (SCFA) content showed that low calcium-phosphorus levels significantly reduced the concentrations of isobutyric acid and isovaleric acids, while high calcium-phosphorus levels reduced the concentration of acetic acid (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB), further confirming that calcium-phosphorus levels in feeds have a significant regulatory effect on intestinal metabolites.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 Linkage between the microbiome and lipidomics contributes to the understanding of calcium-phosphorus-regulated lipid metabolism mechanisms.\u003c/h2\u003e\n\u003cp\u003eBy Spearman's correlation coefficient analysis, we explored the relationship between gut microbial communities and serum glycolipid metabolic indices, which were presented as a heat map (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC). At the genus level, HDL-C showed significant negative correlations with \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, \u003cem\u003eSelenomonas\u003c/em\u003e, and \u003cem\u003eSubdoligranulum\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 to \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas it showed a positive correlation with \u003cem\u003eStreptococcus\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, serum TG showed negative correlation with \u003cem\u003eAlloprevotella\u003c/em\u003e, \u003cem\u003eAnaerovibrio\u003c/em\u003e, \u003cem\u003eMegasphaera\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and positive correlation with \u003cem\u003eLachnospiraceae_AC2044_group\u003c/em\u003e, \u003cem\u003eTerrisporobacter\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Through secreting metabolites into the blood, the gut microbiota is involved in the occurrence and progression of diseases[\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Spearman correlation analysis revealed a correlation between 21 faecal OTUs and 19 significantly different lipid molecules (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). Significantly different lipid molecules TG (18:2_13:0_18:2), Cer (d36:0), and PE (18:1_22:1) correlated with a wide range of differentiated bacterial genera. Among them \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e, which was previously found to be negatively correlated with serum HDL-C concentration, was significantly positively correlated with the lipid molecule Cer (d46:7). \u003cem\u003eAlloprevotella\u003c/em\u003e, which correlated with serum total cholesterol and HDL-C/LDL-C ratio, correlated with TG (18:1_18:1_18:2).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, we systematically investigated for the first time the effects of different calcium and phosphorus levels in the same proportion of feed on lipid metabolism in weaned piglets. It reveals the disorder of lipid metabolism that may result from low calcium-phosphorus levels and elucidates the mechanism of how high-calcium-phosphorus feeds improve lipid metabolism by activating specific signaling pathways. Our results showed that low calcium and phosphorus diets caused significant lipid accumulation in the body and liver of piglets, which was mainly attributed to increased intestinal lipid absorption and abnormal liver lipid accumulation. This finding is consistent with the view of the intestine and liver as the main sites of lipid metabolism and emphasis the importance of regulating the lipid processing capacity of these two organs to maintain lipid homeostasis. Furthermore, our study also showed that by providing a high-calcium phosphate diet, the CAMKK2-AMPKα pathway can be activated, promoting intestinal lipid absorption and expression of transporters, increasing oxidative hydrolysis of renal lipids; At the same time reduce the synthesis of liver lipids, thus effectively improve the lipid metabolic disorder. Combined with the correlation analysis between gut microbial composition and serum lipid metabolism indexes, this study further reveals the important role of gut microbes in the regulation of lipid metabolism. The association of specific microbiota with lipid metabolism-related indicators reinforces the idea that the gut microbiota may be involved in the regulation of lipid metabolism by influencing the secretion patterns of host metabolites. In conclusion, the present study not only highlights the effects of calcium and phosphorus intake on lipid metabolism, but also reveals the potential molecular mechanisms of lipid metabolism regulation, which provides a scientific basis for the development of future nutritional intervention strategies for lipid metabolism disorders. With the further understanding of the mechanism of lipid metabolism, the regulation of calcium and phosphorus intake may become an effective way to improve the disorder of lipid metabolism and prevent related metabolic diseases.\u003c/p\u003e \u003cp\u003eCalcium and phosphorus are not only the most abundant minerals in mammals, but are also involved in a variety of key physiological roles, including lipid metabolism and the synthesis and maintenance of bone structure. As a major energy source and active endocrine organ in the body, lipids play a crucial role in the integrity of cell membranes, protection of vital organs, hormone synthesis, and absorption and transport of vitamins, as well as directly affecting livestock and poultry performance and meat quality[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this study, we found that low calcium and phosphorus diets increased carcass TG and fat percentage, although not significantly, compared with normal calcium and phosphorus levels. This finding echoes previous studies in which calcium supplementation was found to promote muscle fat accumulation[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], whereas phosphorus supplementation showed the opposite effect[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. When calcium and phosphorus were supplemented simultaneously, however, a reduction in muscle lipid accumulation was observed[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which is consistent with our experimental results. Notably, adjustment of calcium and phosphorus levels in the feed significantly affected blood glucose concentrations, where high calcium and phosphorus levels significantly reduced blood glucose levels, suggesting that calcium and phosphorus supplementation may play a role in regulating glucose metabolism. Furthermore, we also observed an effect of calcium and phosphorus supplementation on the HDL-C to LDL-C ratio, consistent with Zhang et al[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Although no significant changes were found in other serum lipid metabolic indexes, the decrease of HDL-C/LDL-C ratio may reflect the changes of lipid metabolism. LDL-C plays a key role in cholesterol transport and the synthesis of cell membranes and certain hormones, while HDL-C is responsible for transporting cholesterol in tissues and maintaining the stability of the cardiovascular system. Therefore, changes in the HDL-C/LDL-C ratio may affect cardiovascular health, suggesting a possible role for calcium and phosphorus in maintaining cardiovascular stability[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the main site of dietary fat absorption, the intestine plays a central role in the overall lipid metabolism. Recent studies have revealed an association between dysregulation of intestinal lipid metabolism and systemic lipid metabolic diseases[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nevertheless, the specific effects of dietary calcium-phosphorus ratios on intestinal lipid metabolism and their underlying mechanisms are not fully understood. The intestinal lipid accumulation, lipid absorption (CD36, FABP2, FABP3, and FABP4), lipid synthesis (DGAT1, DGAT2, ACC, and PPAR γ) gene and protein expression were detected. Here we reveal a correlation between changes in feed calcium and phosphorus levels and intestinal lipid absorption, pointing to a link between enhanced fatty acid uptake capacity and elevated calcium and phosphorus levels. Furthermore, we observed that low calcium and phosphorus levels promote abnormal accumulation of liver lipids, which results from increased lipid synthesis and inhibition of lipid transport and oxidative hydrolysis of key enzyme activities. In contrast, increased calcium and phosphorus levels mitigated this phenomenon, consistent with previous findings that altering calcium or phosphorus levels alone affects lipid accumulation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings emphasis the important influence of the dietary calcium-phosphorus ratio on the lipid metabolism capacity of the intestine-liver axis. Previous studies on rodents[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], poultry[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], fish[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and pigs[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] have all shown that, calcium or phosphorus supplementation alone reduced fat accumulation in the liver and intestines, and we also altered calcium and phosphorus levels in the liver to produce changes consistent with them. The occurrence of this phenomenon may be related to the fact that calcium and phosphorus levels regulate beta oxidation of fatty acids, which is the main pathway of fatty acid degradation. However, the lipid accumulation in the intestine was contrary to previous reports, and the mechanisms need to be further explored because of the paucity of studies on the effects of dietary Ca and P levels on lipid metabolism.\u003c/p\u003e \u003cp\u003eAMPK plays a crucial role in the maintenance of cellular energy homeostasis, not only regulating glycolipid metabolism, but also being involved in the modulation of appetite and anorexia signaling[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Although the inhibitory effect of calcium on endogenous lipid production in the liver by activating the AMPK pathway has been reported, its underlying molecular mechanisms are still at the forefront of exploration[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. CAMKK2, an upstream kinase of AMPK, plays a critical role in regulating lipid metabolism by phosphorylating AMPK at the Thr172 site. Furthermore, the importance of CaMKK2\u0026rsquo;s function in regulating lipid metabolism was demonstrated by the ability of its deletion or pharmacological inhibition to reduce ab initio lipogenesis, which shows potential therapeutic value in ameliorating high-fat diet-induced fatty liver, insulin sensitivity problems[9; 10]. As previously reported, elevated levels of dietary calcium and phosphorus increase intracytoplasmic calcium ion concentrations[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and serum lipocalin concentrations[23; 24], and lipocalin induces activation of the lipocalin receptor 1 (\u003cem\u003eAdipoR1\u003c/em\u003e), which drives CaMKK2 to increase AMPK activity by activating extracellular Ca\u003csup\u003e2+\u003c/sup\u003e efflux[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We further hypothesized that dietary calcium and phosphorus levels might regulate intestinal-liver axis lipid metabolism through the CaMKK2/AMPK signaling pathway. We observed that low calcium-phosphorus levels decreased mRNA and protein levels of intestinal CaMKK2 and AMPKα, accompanied by a decrease in lipid deposition, while high calcium-phosphorus levels reversed this trend. This phenomenon may be related to the upregulation of AMPK activity, which enhances the uptake capacity of long-chain fatty acids (LCFA) in the gut by promoting the expression and membrane translocation of CD36 and FATP4 proteins; This leads to lipid accumulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, we found that dietary calcium and phosphorus supplementation activated the liver CaMKK2/AMPK signaling pathway to mitigate abnormal accumulation of liver lipids by reducing lipid synthesis and enhancing lipid hydrolytic oxidation processes; This process involves the upregulation of SIRT1, a signaling molecule downstream of AMPK. Our results suggest that dietary calcium and phosphorus levels regulate intestinal-liver axis lipid metabolism through the CaMKK2/AMPK signaling pathway.\u003c/p\u003e \u003cp\u003eGiven that Ca and P supplementation is known to influence lipid metabolism in skeletal muscle and adipose tissue[1; 14], the present study examined the effects of changes in calcium and phosphorus dietary levels on liver lipid composition, which is important for understanding the role of calcium and phosphorus in overall metabolic regulation. Our findings reveal that low calcium phosphorus intake is associated with increased levels of triglycerides (MG and TG) and certain phospholipids (PIP\u003csub\u003e2\u003c/sub\u003e and SPH) and sphingolipids (GM3 and SM) in the liver; The concentrations of LPG and Ceramide (Cer) were also reduced. This finding implies that dietary calcium and phosphorus levels are negatively correlated with specific lipid classes in the liver. Through the KEGG pathway analysis of differential lipids, we further verified that these lipids are mainly involved in glyceride and Glycerophospholipid metabolic pathways. The increase of glycerides, especially MG and TG, may reflect the accumulation of intermediate products during lipolysis and the enhancement of activity of TG biosynthesis pathway, this is consistent with existing studies of the effects of calcium and phosphorus supplementation alone on lipid metabolism. In addition, the up-regulation of PIP\u003csub\u003e2\u003c/sub\u003e and SPH, as well as the increase in GM3 and SM, may involve changes in cell signaling, cell proliferation and apoptosis, as well as cell membrane composition and function. In particular, changes in SPH and SM implicate a potential role for sphingolipid metabolism in regulating energy homeostasis and AMPK expression. The changes of SPH and SM may affect the energy metabolism of liver by affecting the physical properties of cell membrane and cell signaling pathway[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In particular, SM (d18:1/16:0) was found to enhance ATP production and reduce AMPK expression by activating glycolytic pathways[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This provides new insights into the interaction between lipid metabolism and energy sensing.\u003c/p\u003e \u003cp\u003eTo establish a potential link between dietary Ca, P, and the microbiome and specific lipid species, we performed a microbiome analysis of colonic contents in the LCAP, HCAP, and CON groups. We found that both the LCAP and HCAP groups increased the alpha diversity of the colonic microbiota compared to the CON group. This may reflect changes in microbial community structure leading to an increase in the diversity of harmful bacteria[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Indeed, a closer examination of the individual bacterial composition of LCAP and HCAP revealed an increased abundance of bacteria belonging to the \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, \u003cem\u003eStreptococcaceae\u003c/em\u003e, and \u003cem\u003eClostridiaceae\u003c/em\u003e, which are families of bacteria that contain pathogenic bacteria [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The β-diversity analysis further revealed significant differences in microbiome structure among the three groups, consistent with previous findings[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our examination of diet-related phylum abundance showed that changes in dietary calcium and phosphorus did not alter the abundance of the \u003cem\u003eFirmicutes\u003c/em\u003e, but the abundance of the \u003cem\u003eBacteroidetes\u003c/em\u003e declined with increasing Ca and P levels, with the \u003cem\u003eFirmicutes\u003c/em\u003e/\u003cem\u003eBacteroidetes\u003c/em\u003e ratio being lowest in the LCAP group. Further analyses of specific ASVs showed that bacteria from the \u003cem\u003ePrevotellaceae\u003c/em\u003e and \u003cem\u003eVeillonellaceae\u003c/em\u003e families decreased with increasing calcium and phosphorus levels, consistent with their association in obesity pathology[32; 33]. Strikingly, the significant reductions in \u003cem\u003eAlloprevotella\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e in the HCAP group compared with the LCAP group were associated with improvements in liver steatosis and other lipid metabolic markers. \u003cem\u003eAlloprevotella\u003c/em\u003e and \u003cem\u003ePrevotella_9\u003c/em\u003e belong to the genus \u003cem\u003eMycobacterium\u003c/em\u003e, which is a group of two in the genus \u003cem\u003ePrevotella\u003c/em\u003e. Several reports have linked \u003cem\u003ePrevotella\u003c/em\u003e to a range of diseases, including advanced liver fibrosis, cirrhosis, insulin resistance, type 2 diabetes, inflammation and obesity[34; 35]. Furthermore, high abundance of \u003cem\u003ePrevotella copri\u003c/em\u003e in a porcine model was associated with elevated concentrations of obesity-related serum metabolites, which were significantly correlated with fat accumulation in pigs[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These results support our observations in this study. Our results provide new insights into the potential role of the gut microbiota in influencing lipid metabolism through calcium and phosphorus levels.\u003c/p\u003e \u003cp\u003eThe endocrine function of the gut plays a key role in the regulation of lipid metabolism, in particular, short-chain fatty acid produced by the fermentation of gut microbes (SCFA) has a significant effect on lipid biosynthesis through the intestine-liver axis[37; 38]. We observed a decreasing trend in total SCFA levels in colonic contents with increasing dietary calcium and phosphorus levels. This finding echoes previous studies, which have shown that the majority of liver lipogenesis is dependent on microbial SCFA from the colon, which are key components of fatty acid biosynthesis[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Further analyses of specific SCFA concentrations revealed significant reductions in acetic acid concentrations in the HCAP group as well as isobutyric and isovaleric acid concentrations in the LCAP group. Acetic acid as the main energy source of the liver, isobutyric acid and isovalerate play critical roles in maintaining intestinal health, providing energy, having anti-inflammatory and immunomodulatory effects, and promoting the integrity of gut barrier function[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Their decline in LCAP seems to be explained by an increase in pathogenic bacteria. Collectively, our results suggest that dietary calcium and phosphorus levels may provide a feasible path to improve liver steatosis and optimize lipid metabolic indices by influencing SCFA production.\u003c/p\u003e \u003cp\u003eIt should be noted that final weight, average daily gain and average daily feed intake are not included in this paper, but they have been assessed and will be part of another paper under consideration elsewhere.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn summary, our results demonstrate that diets low in calcium and phosphorus reduce intestinal lipid absorption and concurrently lead to abnormal accumulation of lipids in the liver and carcass. Conversely, supplementation with calcium and phosphorus can regulate lipid metabolism by activating the CAMKK2/AMPK pathway, promoting the transport of intestinal lipids and the hydrolytic oxidation of liver lipids, and improving the composition of the intestinal microbiota. Overall, we report on the response of intestinal-liver axis lipid metabolism to dietary calcium and phosphorus levels and its regulatory mechanisms, and reveal how these changes affect liver lipid composition and the characteristics of the intestinal microbiome (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings underscore the importance of precisely managing dietary mineral levels to optimize lipid metabolism and enhance the overall health of animals. Furthermore, given the current lack of approved pharmacological treatments for non-alcoholic fatty liver disease (NAFLD), understanding the mechanisms of action of natural mineral elements provides further insights for the development of drugs to prevent and treat NAFLD\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"6 Abbreviations","content":"\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eACC1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eAcetyl-coa Carboxylase 1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eAMPK\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003e5\u0026prime;-Adenosine monophosphate-activated protein kinase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eAMPK\u0026alpha;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eAMP-activated protein kinase alpha\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eCa\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eCalcium\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eCAMKK2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eCalcium/calmodulin-dependent protein kinase kinase 2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eCD36\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eCluster of Differentiation 36\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eCer\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eCeramide\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eCPT1A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eCarnitine palmitoyltransferase 1a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eDGAT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eDiacylglycerol O-acyltransferase 1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eFABP2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eFatty acid binding protein 2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eFABP3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eFatty acid binding protein 3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eFASN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eFatty acid synthase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eFATP4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eFatty acid transport protein 4\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eGLU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eGlucose\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eGM3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eMonosyalilated ganglioside M3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eHDL-C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eHigh-density lipoprotein cholesterol\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eLDL-C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eLow-density lipoprotein cholesterol\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eLPG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eLysophosphatidylglycerol\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eMG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eMonoradylglycerols\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eNAFLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eNonalcoholic fatty liver disease\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eOTU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eOperational taxonomic unit\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003ePhosphorus\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eP-AMPK\u0026alpha;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003ePhosphorylated AMPK\u0026alpha;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eP-CAMKK2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003ePhosphorylated CAMKK2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003ePIP\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003ePhosphatidylinositol 4,5-bisphosphate\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003ePPAR\u0026gamma;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003ePeroxisome proliferator-activated receptor \u0026gamma;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003ePRDM16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003ePR domain-containing protein 16\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSCD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eStearoyl-CoA Desaturase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSCFA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eShort chain fatty acid\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSIRT1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eSirtuin 1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eSphingomyelin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSPH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eSphingosine\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSREPB1C\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eSterol regulatory element-binding protein 1c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eSTTD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eStandardized total tract digestibility\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eTC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eTotal cholesterol\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"226\"\u003e\n\u003cp\u003eTG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"423\"\u003e\n\u003cp\u003eTriglyceride\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the researchers in our laboratory for their efforts and all the staff at the Baiyun Teaching Experimental Base of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences for their selfless support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.M.: Conceptualization, Investigation, Methodology, Data curation, Project administration, Visualization, Writing-original draft. Y.S.: Data curation, Visualization, Writing-original draft. Z.F.: Investigation, Investigation, Data curation. Q.W., X.Y., L.W.: Data curation, Visualization, Writing-original draft. Y.L., Z.J.: Investigation, Methodology, Data curation. H.Y.: Writing-review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the National Key Research and Development Program of China (2021YFD1300402), Research Fund of Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture (No. 2022ZD003), the earmarked fund for China Agriculture Research System (CARS-35), and Special Project for Rural Revitalization Strategy in Guangdong Province (2023TS-3-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were carried out in accordance with the guidelines for the care and use of experimental animals of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences, and approved by the Animal Care Advisory Committee of the Institute of Animal Sciences, Guangdong Academy of Agricultural Sciences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHongbo Yi and Ying Li contributed equally to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGrundmann SM, Ress K, Zimmermann L, Horing M, Liebisch G, Most E et al. 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Gut. 2019;68(2): 359-70. doi:10.1136/gutjnl-2018-316307.\u003c/li\u003e\n\u003cli\u003eYin Y, Sichler A, Ecker J, Laschinger M, Liebisch G, Horing M et al. Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis. J Hepatol. 2023;78(4): 820-35. doi:10.1016/j.jhep.2022.12.028.\u003c/li\u003e\n\u003cli\u003eKindt A, Liebisch G, Clavel T, Haller D, Hormannsperger G, Yoon H et al. The gut microbiota promotes hepatic fatty acid desaturation and elongation in mice. Nat Commun. 2018;9(1): 3760. doi:10.1038/s41467-018-05767-4.\u003c/li\u003e\n\u003cli\u003eCanfora EE, Jocken JW, Blaak EE. Short-chain fatty acids in control of body weight and insulin sensitivity. Nat Rev Endocrinol. 2015;11(10): 577-91. doi:10.1038/nrendo.2015.128.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dietary calcium and phosphorus, Lipid metabolism, Intestine-liver axis, CAMKK2, AMPK","lastPublishedDoi":"10.21203/rs.3.rs-4069024/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4069024/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn the realm of swine production, optimizing body composition and reducing excessive fat accumulation is critical for enhancing both economic efficiency and meat quality. Despite the acknowledged impact of dietary calcium (Ca) and phosphorus (P) on lipid metabolism, the precise mechanisms behind their synergistic effects on fat metabolism remain elusive.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eResearch observations have shown a decreasing trend in the percentage of crude fat in carcasses with increased calcium and phosphorus content in feed. Concurrently, serum glucose concentrations significantly decreased, though differences in other lipid metabolism-related indicators were not significant across groups. Under conditions of low calcium and phosphorus, there is a significant suppression in the expression of FABPs, CD36 and PPARγ in the jejunum and ileum, leading to inhibited intestinal lipid absorption. Concurrently, this results in a marked increase in lipid accumulation in the liver. Conversely, higher levels of dietary calcium and phosphorus promoted intestinal lipid absorption and reduced liver lipid accumulation, with these changes being facilitated through the activation of the CAMKK2/AMPK signaling pathway by high-calcium-phosphorus diets. Additionally, the levels of calcium and phosphorus in the diet significantly altered the composition of liver lipids and the gut microbiota, increasing α-diversity and affecting the abundance of specific bacterial families related to lipid metabolism.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe evidence we provide indicates that the levels of calcium and phosphorus in the diet alter body fat content and lipid metabolism by modulating the response of the gut-liver axis to lipids. These effects are closely associated with the activation of the CAMKK2/AMPK signaling pathway.\u003c/p\u003e","manuscriptTitle":"CAMKK2-AMPK axis endows dietary calcium and phosphorus levels with regulatory effects on lipid metabolism in weaned piglets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 19:02:19","doi":"10.21203/rs.3.rs-4069024/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-04-25T00:47:21+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-02T23:08:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-18T03:03:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-12T11:48:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Animal Science and Biotechnology","date":"2024-03-12T04:01:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-animal-science-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jasb","sideBox":"Learn more about [Journal of Animal Science and Biotechnology](http://jasbsci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jasb/default.aspx","title":"Journal of Animal Science and Biotechnology","twitterHandle":"@animalplantsci","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f4cbe58-d3d0-4a75-aebd-eea221685526","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:06:44+00:00","versionOfRecord":{"articleIdentity":"rs-4069024","link":"https://doi.org/10.1186/s40104-024-01061-0","journal":{"identity":"journal-of-animal-science-and-biotechnology","isVorOnly":false,"title":"Journal of Animal Science and Biotechnology"},"publishedOn":"2024-08-05 15:58:12","publishedOnDateReadable":"August 5th, 2024"},"versionCreatedAt":"2024-03-20 19:02:19","video":"","vorDoi":"10.1186/s40104-024-01061-0","vorDoiUrl":"https://doi.org/10.1186/s40104-024-01061-0","workflowStages":[]},"version":"v1","identity":"rs-4069024","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4069024","identity":"rs-4069024","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}