Association analysis of transcriptome and quasi-targeted metabolomics reveals the regulation mechanism underlying broiler muscle tissue development at different levels of dietary guanidinoacetic acid.

The development and characteristics of muscle fibers in broilers are critical determinants that influence their growth performance, as well as serve as essential prerequisites for the production of high-quality chicken meat. Guanidinoacetic acid (GAA) is a crucial endogenous substance in animal creatine synthesis, and its utilization as a feed additive has been demonstrated the capabilities to enhance animal performance, optimize muscle yield, and augment carcass quality. The objective of this study was to investigate the regulation and molecular mechanism underlying muscle development in broilers at different levels of GAA via multiple omics analysis. The 90 Cobb broilers, aged 1 day, were randomly allocated into three treatments consisting of five replicates of six chickens each. The control group was provided with a basal diet, while the Normal GAA and High GAA groups received a basal diet supplemented with 1.2 g/kg and 3.6 g/kg of GAA, respectively. After a feeding period of 42 days, the pectoralis muscles were collected for histomorphological observation, transcriptome and metabolomic analysis. The results demonstrated that the addition of 1.2 g/kg GAA in the diet led to an augmentation in muscle fiber diameter and up-regulation of IGF1, IHH, ASB2, and ANKRD2 gene expression. However, a high dose of 3.6 g/kg GAA in the diet potentially reversed the beneficial effects on chicken breast development by excessively activating the TGF-ß signaling pathway and reducing nucleotide metabolite content. These findings would provide a theoretical foundation for enhancing the performance and meat quality of broilers by incorporating GAA as a feed additive.

Analysis of microRNA Expression Profiles in Broiler Muscle Tissues by Feeding Different Levels of Guanidinoacetic Acid.

The aim of this study was to explore the molecular mechanisms through which different levels of GAA affect chicken muscle development by influencing miRNA expression, to lay a theoretical foundation for the identification of key functional small RNAs related to poultry muscle development, and to provide new insights into the regulatory mechanisms of GAA on muscle development and meat quality in broilers. It provides a new theoretical basis for using GAA as a feed additive to improve feed performance. Small RNA sequencing technology was utilized to obtain the expression profiles of miRNA in the broiler pectoral muscle fed with different levels of GAA (0 g/kg, 1.2 g/kg and 3.6 g/kg). An analysis of differentially expressed miRNAs revealed 90 such miRNAs in the three combination comparisons, with gga-miR-130b-5p exhibiting significant differences across all three combinations. Furthermore, three of the differentially expressed miRNAs were performed by RT-qPCR verification, yielding results consistent with those obtained from small RNA sequencing. Target gene prediction, as well as the GO and KEGG enrichment analysis of differentially expressed miRNAs, indicated their involvement in muscle cell differentiation and other processes, particularly those associated with the MAPK signaling pathway. This study has, thus, provided valuable insights and resources for the further exploration of the miRNA molecular mechanism underlying the influence of guanidine acetic acid on broiler muscle development. Combined with previous studies and small RNA sequencing, adding 1.2 g/kg GAA to the diet can better promote the muscle development of broilers.

Multiple omics analysis reveals the regulation of SIRT5 on mitochondrial function and lipid metabolism during the differentiation of bovine preadipocytes.

Preadipocyte differentiation represents a critical stage in adipogenesis, with mitochondria playing an undeniable pivotal role. Given the intricate interplay between transcription and metabolic signaling during adipogenesis, the regulation of sirtuin 5 (SIRT5) on mitochondrial function and lipid metabolism was revealed via multiple omics analysis. The findings suggest that SIRT5 plays a crucial role in promoting mitochondrial biosynthesis and maintaining mitochondrial function during preadipocyte differentiation. Moreover, SIRT5 modulates the metabolic levels of numerous bioactive substances by extensively regulating genes expression associated with differentiation, energy metabolism, lipid synthesis, and mitochondrial function. Finally, SIRT5 was found to suppress triacylglycerols (TAG) accumulation while enhancing the proportion and diversity of unsaturated fatty acids, and providing conditions for the expansion and stability of membrane structure during mitochondrial biosynthesis through numerous gene regulations. Our findings provide a foundation for the identification of crucial functional genes, signaling pathways, and metabolic substances associated with adipose tissue differentiation and metabolism.

Integrative analysis of transcriptomics and metabolomics to reveal the melanogenesis pathway of muscle and related meat characters in Wuliangshan black-boned chickens.

BACKGROUND: Melanin is an important antioxidant in food and has been used in medicine and cosmetology. Chicken meat with high melanin content from black-boned chickens have been considered a high nutritious food with potential medicinal properties. The molecular mechanism of melanogenesis of skeletal muscle in black-boned chickens remain poorly understood. This study investigated the biological gene-metabolite associations regulating the muscle melanogenesis pathways in Wuliangshan black-boned chickens with two normal boned chicken breeds as control. RESULTS: We identified 25 differentially expressed genes and 11 transcription factors in the melanogenesis pathways. High levels of the meat flavor compounds inosine monophosphate, hypoxanthine, lysophospholipid, hydroxyoctadecadienoic acid, and nicotinamide mononucleotide were found in Wuliangshan black-boned chickens. CONCLUSION: Integrative analysis of transcriptomics and metabolomics revealed the dual physiological functions of the PDZK1 gene, involved in pigmentation and/or melanogenesis and regulating the phospholipid signaling processes in muscle of black boned chickens.

Hoxa5 alleviates obesity-induced chronic inflammation by reducing ER stress and promoting M2 macrophage polarization in mouse adipose tissue.

Obesity-induced chronic inflammation is associated with endoplasmic reticulum stress (ERS) in adipocytes and changes in both the number and phenotype of adipose tissue macrophages (ATMs). In addition, ERS enhances macrophage activation. So far, the function of Hoxa5 in obesity-induced chronic inflammation has been poorly understood. Herein, we demonstrate the importance of the transcription factor, Hoxa5, in determining adipose tissue macrophage (ATM) polarity and ERS. Hoxa5 decreased bodyweight, reduced inflammatory cytokine secretion and corresponded with an increased number of M2 macrophages in the adipose tissue of high-fat diet (HFD) mice. Transcriptome sequencing data showed that overexpression of Hoxa5 in adipocytes changed expression of endoplasmic reticulum (ER) protein processing-related genes. Based on transcriptome sequencing data and bioinformatics prediction, we have been suggested that Hoxa5 alleviated inflammatory responses by inhibiting ERS and by activating PPARγ pathway in mouse adipose tissue. Hoxa5 alleviated ERS and inflammatory responses by inhibiting the eIF2α/PERK signalling pathway in adipocytes. Hoxa5 also inhibited chronic inflammation of adipocytes by promoting M2 macrophage polarization. In addition, Hoxa5 transcriptionally activated the PPARγ pathway to promote polarization of M2 macrophages, which in turn alleviated chronic inflammation of adipocytes. Taken together, these results shed light on the mechanisms underlying Hoxa5-dependent inhibition of obesity-induced chronic inflammation by reducing ERS and promoting polarization of M2 macrophages. These results suggest that Hoxa5 may be a potential therapeutic target for obesity and other metabolic syndromes.

Homeobox a5 Promotes White Adipose Tissue Browning Through Inhibition of the Tenascin C/Toll-Like Receptor 4/Nuclear Factor Kappa B Inflammatory Signaling in Mice.

Lipopolysaccharide (LPS) induces rapid increase in systemic inflammatory factors. As adipose tissue is a key contributor to the inflammatory response to numerous metabolic stimuli, it is important to understand the mechanism behind the LPS-induced inflammation in white adipose tissue (WAT). Homeobox a5 (Hoxa5) is an important transcription factor, which is highly expressed in adipose tissue, and its mRNA expression is increased at cold exposure in mice. So far, the function of Hoxa5 in adipose tissue browning has been poorly understood. So, the objective of this study was conducted to determine the role of Hoxa5 in adipose inflammatory response and white adipose browning in mice. LPS-induced inflammatory and cold-induced browning model were conducted. We compared the coordinated role of Hoxa5 in inflammation and thermogenesis of mice adipose. Transcriptional and methylation regulation was determined by luciferase assay, electrophoretic mobility shift assay, and bisulfite conversion experiment. Hoxa5 and tenascin C (TNC) were involved in WAT inflammation and browning in mice with LPS injection. Furthermore, Hoxa5 inhibited the TNC-involved activation of Toll-like receptor (TLR) 4/nuclear factor kappa B (NF-κB) signal pathway and promoted WAT browning. Moreover, we found that a BMP4/Smad1 signal, closely related to browning, was activated by Hoxa5. Hoxa5 relieved adipocyte inflammation by decreasing TNC-mediated TLR4 transducer and activator of the NF-κB pathway. Interestingly, descended methylation level increased Hoxa5 expression in cold exposure. Our findings demonstrated that Hoxa5 alleviated inflammation and enhanced browning of adipose tissue via negative control of TNC/TLR4/NF-κB inflammatory signaling and activating BMP4/Smad1 pathway. These findings indicated a novel potential means for the regulation of inflammation in adipocytes to prevent obesity and other inflammatory diseases.

Hoxa5 Promotes Adipose Differentiation via Increasing DNA Methylation Level and Inhibiting PKA/HSL Signal Pathway in Mice.

BACKGROUND/AIMS: Impaired adipogenesis may be the underlying cause in the development of obesity and type II diabetes. Mechanistically, the family of Homeobox transcription factors is implicated in the regulation of adipocyte fate. Hoxa5 is highly expressed in adipocytes, and its mRNA expression is decreased during differentiation. However, the function of Hoxa5 in adipose tissue has been poorly understood. The aim of this study is to unveil the role of Hoxa5 on adipocyte differentiation and its underlying mechanisms. METHODS: Quantitative real-time PCR (qPCR) and western blot were performed to determine Hoxa5 expression in primary adipocytes and in adipose tissues from mice. Lipid accumulation was evaluated by bodipy staining. Dual luciferase assay was applied to explore the transcription factor of Hoxa5 and the transcriptional target gene modulated by Hoxa5. All measurements were performed at least for three times at least. RESULTS: A significant reduction of Hoxa5 expression was observed in adipose tissue of High Fat Diet (HFD) induced obesity mice. We determined Hoxa5 increased adipocytes differentiation and mitochondrial biogenesis in adipocytes in vitro. CEBPß was determined a transcription factor of Hoxa5 and inhibited methylation level of Hoxa5 by combining on the promoter of Hoxa5. Importantly, we found Fabp4, a known positive regulator of adipocytes differentiation, was transcriptional activation by Hoxa5. In addition, Hoxa5 promotes adipocytes differentiation by inhibiting PKA/HSL pathway. CONCLUSION: Our study demonstrated the promoting role of Hoxa5 in adipocytes differentiation and therefore bringing a new therapeutic mean to the treatment of obesity and type II diabetes.

αMSH prevents ROS-induced apoptosis by inhibiting Foxo1/mTORC2 in mice adipose tissue.

Alpha-melanocyte stimulating hormone (αMSH) is an important adenohypophysis polypeptide hormone that regulates body metabolic status. To date, it is well known that the disorder of hypothalamic αMSH secretion is related to many metabolic diseases, such as obesity and type II diabetes. However, the underlying mechanisms are poorly understood. In our study, we focused on the reactive oxygen species (ROS)-induced adipocyte apoptosis and tried to unveil the role of αMSH in this process and the signal pathway which αMSH acts through. Kunming white mice were used and induced to oxidative stress status by hydrogen peroxide (H2O2) injection and a significant reduction of αMSH were found in mice serum, while elevated ROS level and mRNA level of pro-apoptotic genes were observed in mice adipose tissue. What is more, when detect the function of αMSH in ROS-induced apoptosis, similar inhibitory trend was found with the oxidative stress inhibitor N-acetyl-L-cysteine (NAC) in ROS-induced adipocyte apoptosis and this trend is αMSH receptor melanocortin 5 receptor (MC5R) depended, while an opposite trend was found between αMSH and Foxo1, which is a known positive regulator of adipocyte apoptosis. Further, we found that the repress effect of αMSH in adipocytes apoptosis is acting through Foxo1/mTORC2 pathway. These findings indicate that, αMSH has a strong inhibitory effect on ROS-induced adipocyte apoptosis and underlying mechanism is interacting with key factors in mTOR signal pathway. Our study demonstrated a great role of αMSH in adipocyte apoptosis and brings a new therapeutic mean to the treatment of obesity and diabetes.

Mark4 promotes oxidative stress and inflammation via binding to PPARγ and activating NF-κB pathway in mice adipocytes.

MAP/Microtubule affinity-regulating kinase 4 (Mark4) plays an important role in the regulation of microtubule organization, adipogenesis and apoptosis. However, the role of Mark4 plays in oxidative stress and inflammation are poorly understood. In this study, we found Mark4 was induced by high fat diet (HFD) while PPARγ was elevated significantly in mice adipocytes. Further analyses revealed Mark4 impaired mitochondrial oxidative respiration and increased reactive oxygen species (ROS) production. At same time, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were greatly reduced. By treating cells with H2O2 and vitamin E (VE), Mark4 accentuated oxidative stress along with increased mRNA level of inflammatory factor interleukin-6 (IL-6) and decreased leptin mRNA. Furthermore, we found PPARγ bind to Mark4 promoter region and inhibited Mark4 expression. We showed PPARγ interacted with Mark4 and inhibited the stimulating effect of Mark4 on oxidative stress and inflammation. Finally, we demonstrated that the IKKα/NF-κB signal pathway was involved in Mark4 induced oxidative stress and inflammation, while PTDC, a special inhibitor of NF-κB signal pathway, reduced oxidative stress and inflammation. Thus, our study indicated that Mark4 was a potential drug target for treating metabolic diseases.

FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes.

Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process.